U.S. patent number 8,192,944 [Application Number 13/137,257] was granted by the patent office on 2012-06-05 for protein fragment complementation assays for the detection of biological or drug interactions.
This patent grant is currently assigned to Odyssey Thera Inc.. Invention is credited to Stephen William Watson Michnick, Joelle Nina Polletier, Ingrid Remy.
United States Patent |
8,192,944 |
Michnick , et al. |
June 5, 2012 |
Protein fragment complementation assays for the detection of
biological or drug interactions
Abstract
We describe a strategy for designing and implementing
protein-fragment complementation assays (PCAs) to detect
biomolecular interactions in vivo and in vitro. The design,
implementation and broad applications of this strategy are
illustrated with a large number of enzymes with particular detail
provided for the example of murine dihydrofolate reductase (DHFR).
Fusion peptides consisting of N- and C-terminal fragments of murine
DHFR fused to GCN4 leucine zipper sequences were coexpressed in
Escherichia coli grown in minimal medium, where the endogenous DHFR
activity was inhibited with trimethoprim. Coexpression of the
complementary fusion products restored colony formation. Survival
only occurred when both DHFR fragments were present and contained
leucine-zipper forming sequences, demonstrating that reconstitution
of enzyme activity requires assistance of leucine zipper formation.
DHFR fragment-interface point mutants of increasing severity (Ile
to Val, Ala and Gly) resulted in a sequential increase in E. coli
doubling times illustrating the successful DHFR fragment reassembly
rather than non-specific interactions between fragments. This assay
could be used to study equilibrium and kinetic aspects of molecular
interactions including protein-protein, protein-DNA, protein-RNA,
protein-carbohydrate and protein-small molecule interactions, for
screening cDNA libraries for binding of a target protein with
unknown proteins or libraries of small organic molecules for
biological activity. The selection and design criteria applied here
is developed for numerous examples of clonal selection,
colorometric, fluorometric and other assays based on enzymes whose
products can be measured. The development of such assay systems is
shown to be simple, and provides for a diverse set of protein
fragment complementation applications.
Inventors: |
Michnick; Stephen William
Watson (Westmount, CA), Polletier; Joelle Nina
(Westmount, CA), Remy; Ingrid (Montreal,
CA) |
Assignee: |
Odyssey Thera Inc. (San Ramon,
CA)
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Family
ID: |
4159802 |
Appl.
No.: |
13/137,257 |
Filed: |
August 1, 2011 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20110287950 A1 |
Nov 24, 2011 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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11650466 |
Jan 8, 2007 |
7989218 |
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10353090 |
Jan 29, 2003 |
7160691 |
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10154758 |
May 24, 2002 |
6929916 |
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09499464 |
Feb 7, 2000 |
6428951 |
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09017412 |
Feb 2, 1998 |
6270964 |
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Foreign Application Priority Data
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Jan 31, 1997 [CA] |
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2196496 |
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Current U.S.
Class: |
435/7.1;
436/518 |
Current CPC
Class: |
C07K
14/43595 (20130101); G01N 33/5008 (20130101); G01N
33/542 (20130101); G01N 33/573 (20130101); C12N
15/1055 (20130101); G01N 33/58 (20130101); G01N
33/68 (20130101); G01N 33/6845 (20130101); A01K
67/0271 (20130101); G01N 33/6803 (20130101); G01N
33/581 (20130101); A01K 2267/0331 (20130101); Y02A
90/10 (20180101); C07K 2319/00 (20130101); Y02A
90/26 (20180101); A01K 2267/0393 (20130101) |
Current International
Class: |
G01N
33/53 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
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6270964 |
August 2001 |
Michnick et al. |
6428951 |
August 2002 |
Michnick et al. |
7160691 |
January 2007 |
Michnick et al. |
|
Primary Examiner: Cheu; Jacob
Attorney, Agent or Firm: Angres; Isaac A.
Parent Case Text
This application is a continuation of application Ser. No.
11/650,466 filed Jan. 8, 2007, now U.S. Pat. No. 7,989,218; which
application is a continuation of U.S. Ser. No. 10/353,090 filed
Jan. 29, 2003, now U.S. Pat. No. 7,160,691; which application is a
continuation of U.S. Ser. No. 10/154,758 filed May 24, 2002, now
U.S. Pat. No. 6,929,916; which application is a continuation
09/499,464 filed Feb. 7, 2000; now U.S. Pat. No. 6,428,951; which
application is a continuation of U.S. Ser. No. 09/017,412 filed
Feb. 2, 1998, now U.S. Pat. No. 6,270,964.
Claims
What is claimed is:
1. Molecular fragment complementation assays for the detection of
molecular interactions comprising a reassembly of separate
fragments of a molecule, wherein reassembly of said fragments is
operated by the interaction of molecular domains fused to each
fragment of said molecules, and wherein reassembly of the fragments
is independent of other molecular processes.
2. A composition comprising a complementary fragment of a first
protein molecule which exhibits a detectable activity when
associated, wherein each fragment is fused to a separate protein
molecule.
Description
FIELD OF THE INVENTION
The present invention relates to the determination of the function
of novel gene products. The invention further relates to Protein
fragment Complementation Assays (PCA). PCAs allow for the detection
of a wide variety of types of protein-protein, protein-RNA,
protein-DNA, Protein-carbohydrate or protein-small organic molecule
interactions in different cellular contexts appropriate to the
study of such interactions.
BACKGROUND OF THE INVENTION
Many processes in biology, including transcription, translation,
and metabolic or is signal transduction pathways, are mediated by
noN-covalently-associated multienzyme complexes.sup.1, 101. The
formation of multiprotein or protein-nucleic acid complexes produce
the most efficient chemical machinery. Much of modern biological
research is concerned with identifying proteins involved in
cellular processes, determining their functions and how, when, and
where they interact with other proteins involved in specific
pathways. Further, with rapid advances in genome sequencing
projects there is a need to develop strategies to define "protein
linkage maps", detailed inventories of protein interactions that
make up functional assemblies of proteins.sup.2,3. Despite the
importance of understanding protein assembly in biological
processes, there are few convenient methods for studying
protein-protein interactions in vivo.sup.4,5. Approaches include
the use of chemical crosslinking reagents and resonance energy
transfer between dye-coupled proteins.sup.102, 103. A powerful and
commonly used strategy, the yeast two-hybrid system, is used to
identify novel protein-protein interactions and to examine the
amino acid determinants of specific protein interactions.sup.4,6-8.
The approach allows for rapid screening of a large number of
clones, including cDNA libraries. Limitations of this technique
include the fact that the interaction must occur in a specific
context (the nucleus of S. cerevisiae), and generally cannot be
used to distinguish induced versus constitutive interactions.
Recently, a novel strategy for detecting protein-protein
interactions has been demonstrated by Johnsson and
Varshaysky.sup.108 called the ubiquitin-based split protein sensor
(USPS).sup.9. The strategy is based on cleavage of proteins with
N-terminal fusions to ubiquitin by cytosolic proteases
(ubiquitinases) that recognize its tertiary structure. The strategy
depends on the reassembly of the tertiary structure of the protein
ubiquitin from complementary N- and C-terminal fragments and
crucially, on the augmentation of this reassembly by
oligomerization domains fused to these fragments. Reassembly is
detected as specific proteolysis of the assembled product by
cytosolic proteases (ubiquitinases). The authors demonstrated that
a fusion of a reporter protein-ubiquitin C-terminal fragment could
also be cleaved by ubiquitinases, but only if co-expressed with an
N-terminal fragment of ubiquitin that was complementary to the
C-terminal fragment. The reconstitution of observable ubiquitinase
activity only occurred if the N- and C-terminal fragments were
bound through GCN4 leucine zippers.sup.109,110. The authors
suggested that this "split-gene" strategy could be used as an in
vivo assay of protein-protein interactions and analysis of protein
assembly kinetics in cells. Unfortunately, this strategy requires
additional cellular factors (in this case ubiquitinases) and the
detection method does not lend itself to high-throughput screening
of cDNA libraries.
Rossi, F., C. A. Charlton, and H. M. Blau (1997) Proc. Nat. Acad.
Sci. (USA) 94, 8405-8410) have reported an assay based on the
classical complementation of .alpha. and .omega. fragments of
.beta.-galactosidase (.beta.-gal) and induction of complementation
by induced oligomerization of the proteins FKBP12 and the mammalian
target of rapamycin by rapamycin in transfected C2C12 myoblast cell
lines. Reconstitution of b-gal activity is detected using substrate
fluorescein di-.beta.-D-galactopyranoside using several
fluorescence detection assays. While this assay bears some
resemblance to the present invention, there are several significant
distinguishing differences. First, this particular complementation
approach has been used for over thirty years in a vast number of
applications including the detection of protein-protein
interactions. Krevolin, M. and D. Kates (1993) U.S. Pat. No.
5,362,625) teaches the use of this complementation to detect
protein-protein interactions. Also achievement of .beta.-gal
complementation in mammalian cells has previously been reported
(Moosmann, P. and S. Rusconi (1996) Nucl. Acids Res. 24,
1171-1172). The individual PCAs presented here are completely de
novo designed interaction detection assays, not described in any
way previously except for publications arising from applicants
laboratory. Secondly, this application describes a general strategy
to develop molecular interaction assays from a large number of
enzyme or protein detectors, all de novo designed assays, whereas
the .beta.-gal assay is not novel, nor are any general strategies
or advancements over previously well documented applications
given.
As in the USPS, the yeast-two hybrid strategy requires additional
cellular machinery for detection that exist only in specific
cellular compartments. There is therefore a need for a detection
system which uses the reconstitution of a specific enzyme activity
from fragments as the assay itself, without the requirement for
other proteins for the detection of the activity. Preferably, the
assay would involve an oligomerization-assisted complementation of
fragments of monomeric or multimeric enzymes that require no other
proteins for the detection of their activity. Furthermore, if the
structure of an enzyme were known it would be possible to design
fragments of the enzyme to ensure that the reassembled fragments
would be active and to introduce mutations to alter the stringency
of detection of reassembly. However, knowledge of structure is not
a prerequisite to the design of complementing fragments, as will be
explained below. The flexibility allowed in the design of such an
approach would make it applicable to situations where other
detection systems may not be suitable.
Recent advances in human genomics research has led to rapid
progress in the identification of novel genes. In applications to
biological and pharmaceutical research, there is now the pressing
need to determine the functions of novel gene products; for
example, for genes shown to be involved in disease phenotypes. It
is in addressing questions of function where genomics-based
pharmaceutical research becomes bogged down and there is now the
need for advances in the development of simple and automatable
functional assays. A first step in defining the function of a novel
gene is to determine its interactions with other gene products in
an appropriate context; that is, since proteins make specific
interactions with other proteins or other biopolymers as part of
functional assemblies, an appropriate way to examine the function
of a novel gene is to determine its physical relationships with the
products of other genes.
Screening techniques for protein interactions, such as the yeast
"two-hybrid" system, have transformed molecular biology, but can
only be used to study specific types of constitutively interacting
proteins or interactions of proteins with other molecules, in
narrowly defined cellular and compartmental contexts and require a
complex cellular machinery (transcription) to work. To rationally
screen for protein interactions within the context of a specific
problem requires more flexible approaches. Specifically, assays
that meet criteria necessary not only to detecting molecular
interactions, but also to validating these interactions as specific
and biologically relevant.
A list of assay characteristics that meet such criteria are as
follows:
1) Allow for the detection of protein-protein, protein-DNA/RNA or
protein-drug interactions in vivo or in vitro.
2) Allow for the detection of these interactions in appropriate
contexts, such as within a specific organism, cell type, cellular
compartment, or organelle.
3) Allow for the detection of induced versus constitutive
protein-protein interactions (such as by a cell growth or
inhibitory factor).
4) To be able to distinguish specific versus non-specific
protein-protein interactions by controlling the sensitivity of the
assay.
5) Allow for the detection of the kinetics of protein assembly in
cells.
6) Allow for screening of cDNA, small organic molecule, or DNA or
RNA libraries for molecular interactions.
SUMMARY OF THE INVENTION
The present invention seeks to provide the above-mentioned needs
for which the prior art is silent. The present invention provides a
general strategy for detecting protein interactions with other
biopolymers including other proteins, nucleic acids, carbohydrates
or for screening small molecule libraries for compounds of
potential therapeutic value. In a preferred embodiment, the instant
invention seeks to provide an oligomerization-assisted
complementation of fragments of monomeric enzymes that require no
other proteins for the detection of their activity. In one such
embodiment, a protein-fragment complementation assay (PCA) based on
reconstitution of dihydrofolate reductase activity by
complementation of defined fragments of the enzyme in E. coli is
hereby provided. This assay requires no additional endogenous
factors for detecting specific protein-protein interactions (i.e.
leucine zipper interactions) and can be conveniently extended to
screening cDNA, nucleic acid, small molecule or protein design
libraries for molecular interactions. In addition, the assay can
also be adapted for detection of protein interactions in any
cellular context or compartment and be used to distinguish between
induced versus constitutive protein interactions in both
prokaryotic and eukaryotic systems.
One particular strategy for designing a protein complementation
assay (PCA) is based on using the following characteristics: 1) A
protein or enzyme that is relatively small and monomeric, 2) for
which there is a large literature of structural and functional
information, 3) for which simple assays exist for the
reconstitution of the protein or activity of the enzyme, both in
vivo and in vitro, and 4) for which overexpression in eukaryotic
and prokaryotic cells has been demonstrated. If these criteria are
met, the structure of the enzyme is used to decide the best
position in the polypeptide chain to split the gene in two, based
on the following criteria: 1) The fragments should result in
subdomains of continuous polypeptide; that is, the resulting
fragments will not disrupt the subdomain structure of the protein,
2) the catalytic and cofactor binding sites should all be contained
in one fragment, and 3) resulting new N- and C-termini should be on
the same face of the protein to avoid the need for long peptide
linkers and allow for studies of orientation-dependence of protein
binding.
It should be understood that the above mentioned criteria do not
all need to be satisfied for a proper working of the present
invention. It is an advantage that the enzyme be small, preferably
between 10-40 kDa. Although monomeric enzymes are preferred,
multimeric enzymes can also be envisaged as within the scope of the
present invention. The dimeric protein tyrosinase can be used in
the instant assay. The information on the structure of the enzyme
provides an additional advantage in designing the PCA, but is not
necessary. Indeed, an additional strategy, to develop PCAs is
presented, based on a combination of exonuclease
digestion-generated protein fragments followed by directed protein
evolution in application to the enzyme aminoglycoside kinase.
Although the overexpression in prokaryotic cells is preferred it is
not a necessity. It will be understood to the skilled artisan that
the enzyme catalytic site (of the chosen enzyme) does not
absolutely need to be on same molecule.
The present application explains the rationale and criteria for
using a particular enzyme in a PCA. FIG. 1 shows a general
description of a PCA. The gene for a protein or enzyme is
rationally dissected into two or more fragments. Using molecular
biology techniques, the chosen fragments are subcloned, and to the
5' ends of each, proteins that either are known or thought to
interact are fused. Co-transfection or transformation these DNA
constructs into cells is then carried out. Reassembly of the probe
protein or enzyme from its fragments is catalyzed by the binding of
the test proteins to each other, and reconstitution is observed
with some assay. It is crucial to understand that these assays will
only work if the fused, interacting proteins catalyze the
reassembly of the enzyme. That is, observation of reconstituted
enzyme activity must be a measure of the interaction of the fused
proteins.
A preferred embodiment of the present invention focuses on a PCA
based on the enzyme dihydrofolate reductase. Expansion of the
strategy to include assays in eukaryotic, cells, library screening,
and a specific application to problems concerning the study of
integrated biochemical pathways such as signal transduction
pathways, is presented. Additional assays, including those based on
enzymes that can act as dominant or recessive drug selection or
metabolic salvage pathways are disclosed. In addition, PCAs based
on enzymes that will produce a colored or fluorescent product are
also disclosed. The present invention teaches how the PCA strategy
can be both generalized and automated for functional testing of
novel genes, screening of natural products or compound libraries
for pharmacological activity and identification of novel gene
products that interact with DNA, RNA or carbohydrates are
disclosed. It also teaches how the PCA strategy can be applied to
identifying natural products or small molecules from compound
libraries of potential therapeutic value that can inhibit or
activate such molecular interactions and how enzyme substrates and
small molecule inhibitors of enzymes can be identified. Finally, it
teaches how the PCA strategy can be used to perform protein
engineering experiments that could lead to designed enzymes with
industrial applications or peptides with biological activity.
Simple strategies to design and implement assays for detecting
protein interactions in vivo are disclosed herein. We have designed
complementary fragments of the native mDHFR that, when coexpressed
in E. coli grown in minimal medium, allow for survival of clones
expressing the two fragments, where the basal activity of the
endogenous bacterial DHFR is inhibited by the competitive inhibitor
trimethoprim (FIG. 3). Reconstitution of activity only occurred
when both N- and C-terminal fragments of DHFR were coexpressed as
C-terminal fusions to GCN4 leucine zipper sequences, indicating
that reassembly of the fragments requires formation of a leucine
zipper between the N- and C-terminal fusion peptides. The
sequential increase in cell doubling times resulting from the
destabilizing mutations directed at the assembly interface (Ile114
to Val, Ala or Gly) demonstrates that the observed cell survival
under selective conditions is a result of the specific,
leucine-zipper-assisted association of mDHFR fragment[1,2] with
fragment[3], as opposed to nonspecific interactions of Z-F[3] with
Z-F[1,2]. several detailed and many additional examples are
given.
As demonstrated previously with the ubiquitin-based split protein
sensor (USPS).sup.9, a protein-fragment complementation strategy
can be used to study equilibrium and kinetic aspects of
protein-protein interactions in vivo. The DHFR and other PCAs
however, are simpler assays. They are complete systems; no
additional endogenous factors are necessary and the results of
complementation are observed directly, with no further
manipulation. The E. coli cell survival assay described herein
should therefore be particularly useful for screening cDNA
libraries for protein-protein interactions. mDHFR expression in
cells can be monitored by binding of fluorescent high-affinity
substrate analogues for DHFR.sup.26.
There are several further aspects of the PCAs that distinguish them
from all other strategies for studying protein-protein interactions
in vivo (except USPS). We have designed complementary fragments of
enzymes that allow for controlling the stringency of the assay, and
could be used to obtain estimates of the kinetics and equilibrium
constants for association of two proteins. For example, with DHFR
the point mutations of the wild-type enzyme Ile 114 to Val, Ala, or
Gly alter the stringency of reconstitution of DHFR activity. For
determining estimates of equilibrium and kinetic parameters for a
specific protein-protein interaction, one could perform a series of
DHFR PCA experiments with two proteins that interact with a known
affinity, using the wild type or destabilizing mutant DHFR
fragments. Comparison of cell growth rates in this model system
with rates for a DHFR PCA using unknowns would give an estimate of
the strength of the unknown interaction.
It should be understood that the present invention should not be
limited to the DHFR or other PCAs presented, as it is only
non-limiting embodiments of the protein complementation assay of
the present invention. Moreover, the PCAs should not be limited in
the context in which they could be used. Constructs could be
designed for targeting the PCA fusions to specific compartments in
the cell by addition of signaling peptide sequences.sup.27,28.
Induced versus constitutive protein-protein interactions could be
distinguished by a eukaryotic version of the PCA, in the case of an
interaction that is triggered by a biochemical event. Also, the
system could be adapted for use in screening for novel, induced
protein-molecular associations between a target protein and an
expression library.
The instant invention is also directed to a method for detecting
biomolecular interactions said method comprising:
(a) selecting an appropriate reporter molecule;
(b) effecting fragmentation of said reporter molecule such that
said fragmentation results in reversible loss of reporter
function;
(c) fusing or attaching fragments of said reporter molecule
separately to other molecules; followed by
(d) reassociation of said reporter fragments through interactions
of the molecules that are fused to said fragments.
The invention also provides molecular fragment complementation
assays for the detection of molecular interactions comprising a
reassembly of separate fragments of a molecule, wherein reassembly
of said fragments is operated by the interaction of molecular
domains fused to each fragment of said molecules, and wherein
reassembly of the fragments is independent of other molecular
processes.
In another aspect, the present invention is directed to a method of
testing biomolecular interactions comprising:
a) generating a first fusion product comprising i) a first fragment
of a first molecule and ii) a second molecule which is different or
the same as said first molecule;
b) generating a second fusion product comprising i) a second
fragment of said first molecule; and ii) a third molecule which is
different from or the same as said first molecule or second
molecule;
c) allowing the first and second fusion products to contact each
other; and
d) testing for activity regained by association of the recombined
fragments of the first molecule, wherein said reassociation is
mediated by interaction of the second and third molecules.
In another novel feature, the invention is directed to a method
comprising an assay where fragments of a first molecule are fused
to a second molecule and fragment association is detected by
reconstitution of the first molecule's activity.
The present invention also provides a composition comprising a
product selected from the group consisting of:
(a) a first fusion product comprising: 1) a first fragment of a
first molecule whose fragments can exhibit a detectable activity
when associated and
2) a second molecule that can bind (a)(1);
(b) a second fusion product comprising 1) a second fragment of said
first molecule and 2) a third molecule that can bind (b)(1);
and
c) both (a) and (b).
The invention further provides a composition comprising
complementary fragments of a first molecule, each fused to a
separate fragment of a second molecule.
The inventors of the present subject matter further provide a
composition comprising a nucleic acid molecule coding for a fusion
product, which molecule comprises sequences coding for a product
selected from the group consisting of:
(a) a first fusion product comprising: 1) fragments of a first
molecule whose fragments can exhibit a detectable activity when
associated and 2) a second molecule fused to the fragment of the
first molecule;
(b) a second fusion product comprising 1) a second fragment of said
first molecule and 2) a second or third molecule; and
(c) both (a) and (b).
The present invention is also directed to a method of testing for
biomolecular interactions associated with: (a) complementary
fragments of a first molecule whose fragments can exhibit a
detectable activity when associated or (b) binding of two
protein-protein interacting domains from a second or third
molecule, said method comprising: 1) creating a fusion of (a) a
first fragment of a first molecule whose fragments can exhibit a
detectable activity when associated and (b) a first protein-protein
interacting domain; 2) creating a fusion of (a) a second fragment
of said first molecule and (b) a second protein-protein interacting
domain that can bind said first protein-protein interacting domain;
3) allowing the fusions of (1) and (2) to contact each other; and
4) testing for said activity.
The instant invention further provides a composition comprising a
product selected from the group consisting of:
(a) a first fusion product comprising: 1) a first fragment of a
molecule whose fragments can exhibit a detectable activity when
associated and 2) a first protein-protein interacting domain;
(b) a second fusion product comprising 1) a second fragment of said
first molecule and 2) a second protein-protein interacting domain
that can bind said first protein-protein interacting domain;
and
(c) both (a) and (b).
The invention is also directed to a composition comprising a
nucleic acid molecule coding for a fusion product, which molecule
comprises sequences coding for either:
(a) a first fusion product comprising: 1) a first fragment of a
molecule whose fragments can exhibit a detectable activity when
associated and 2) a first protein-protein interacting domain;
or
(b) a second fusion product comprising 1) a second fragment of said
molecule and 2) a second protein-protein interacting domain that
can bind said first protein-protein interacting domain; or
(c) both (a) and (b).
The invention also provides a method of detecting kinetics of
protein assembly and screening cDNA libraries comprising performing
PCA.
In another embodiment, the invention further provides a method of
testing the ability of a compound to inhibit molecular interactions
in a PCA comprising performing a PCA in the presence of said
compound and correlating any inhibition with said presence.
In a further embodiment, the invention provides a method for
detecting protein-protein interactions in living organisms and or
cells, which method comprises:
(a) synthesizing probe protein fragments from an enzyme which
enables dominant selection by dissecting the gene coding for the
enzyme into at least two fragments;
(b) constructing fusion proteins with one or more molecules that
are to be tested for interactions;
(c) fusing the proteins obtained in (b) with one or more of the
probe fragments;
(d) coexpressing the fusion proteins; and
(e) detecting the reconstitution of enzyme activity.
The invention still provides a method for detecting biomolecular
interactions said method comprising:
(a) selecting an appropriate reporter molecule;
(b) effecting fragmentation of said reporter molecule;
(c) fusing or attaching fragments of said reporter molecule
separately to other molecules; followed by
(d) reassociation of said reporter fragments through interactions
of the molecules that are fused to said fragments.
Lastly, the invention also provides a novel method of affecting
gene therapy, which includes the step of providing the assays and
compositions described above.
The present invention is pioneering as it is the first protein
complementation assay displaying such a level of simplicity and
versatility. The exemplified embodiments are protein-fragment
complementation assays (PCA) based on mDHFR, where a leucine zipper
directs the reconstitution of DHFR activity. Activity was detected
by an E. coli survival assay which is both practical and
inexpensive. This system illustrates the use of mDHFR fragment
complementation in the detection of leucine zipper dimerization and
could be applied to the detection of unknown, specific
protein-molecular interactions in vivo.
It should be understood that the instant invention is not limited
to the PCAs presented here, as numerous other enzymes can be
selected and used in accordance with the teachings of the present
invention. Examples of such markers can be found in Kaufman, (1987
Genetic Eng. 9:155-198) and references found therein as well as
table 1 of this application.
It should also be clear to the skilled artisan to which the present
invention pertains that the invention is not limited to the use of
leucine zippers as the two interacting molecules. Indeed, numerous
other types of protein-molecule interactions can be used and
identified in accordance with the teaching of the present
invention. The known types of motifs involved in protein-molecular
interactions are well known in the art.
The present application refers to numerous prior art documents and
the entire contents of all those prior art documents are herein
incorporated by reference.
Other features and advantages of the present invention will be
apparent from the following description of the preferred
embodiments thereof, the appended Examples and from the enjoined
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides a general description of a PCA. Using molecular
biology techniques, the chosen fragments of the enzyme are
subcloned, and to the 5' ends of each, proteins that either are
known or thought to interact are fused. Co-transfection or
transformation these DNA constructs into cells is then carried out
and reconstitution with some assay is observed.
FIG. 2 is a scheme of the fusion constructs used in one of the
embodiments of the invention. The hexahistidine peptide (6His), the
homodimerizing GCN4 leucine zipper (Zipper) and mDHFR fragments (1,
2 and 3) are illustrated. The labels for the constructs are used to
identify both the DNA constructs and the proteins expressed from
these constructs.
FIG. 3: (A) shows E. coli survival assay on minimal medium plates.
Control: Left side of the plate: E. coli harboring pQE-30 (no
insert); right side: E. coli harboring pQE-16, coding for native
mDHFR. Panel I: Left side of each plate: transformation with
construct Z-F[1,2]; right side of each plate: transformation with
construct Z-F[3]. Panel II: Cotransformation with constructs
Z-F[1,2] and Z-F[3]. Panel III: Cotransformation with constructs
Control-F[1,2] and Z-F[3]. All plates contain 0.5 mg/ml
trimethoprim. In panels I to III, plates on the right side contain
1 mM IPTG.
(B) E. coli survival assay using destabilizing DHFR mutants. Panel
I: Cotransformation of E. coli with constructs Z-F[1,2] and
Z-F[3:Ile114Val]. Panel II: Cotransformation with Z-F[1,2] and
Z-F[3:Ile114Ala]. Inset is a 5-fold enlargement of the right-side
plate. Panel III: Cotransformation with Z-F[1,2] and
Z-F[3:Ile114Gly]. All plates contain 0.5 mg/ml trimethoprim. Plates
on the right side contain 1 mM IPTG.
FIG. 4 features the coexpression of mDHFR fragments. (A) Agarose
gel analysis of restriction pattern resulting from HincII digestion
of plasmid DNA. Lane 1 contains DNA isolated from E. coli
cotransformed with constructs Z-F[1,2] and Z-F[3]. Lanes 2 and 3
contain DNA isolated from E. coli transformed with, respectively,
construct Z-F[3] and construct Z-F[1,2]. Fragment migration (in bp)
is indicated to the right.
(B) SDS-PAGE analysis of mDHFR fragment expression. Lanes 1 to 5
show crude lysate of untransformed E. coli (lane 1), or E. coli
expressing Z-F[1,2] (20.8 kDa; lane 2), Z-F[3] (18.4 kDa; lane 3),
Control-F[1,2] (14.2 kDa; lane 4), and Z-F[1,2]+Z-F[3] (lane 5).
Lane 6 shows 40 ml out of 2 ml copurified Z-F[1,2] and Z-F[3].
Arrowheads point to the proteins of interest. Migration of
molecular weight markers (in kDa) is indicated to the right.
FIG. 5 illustrates the general features of a PCA based on a
survival assay such as the DHFR PCA. The assay can be used in a
bacterial or a mammalian context. The inserted target DNA can be a
known sequence coding for a protein (or protein domain) of
interest, or can be a cDNA library.
FIG. 6 represents an autoradiograph of a COS cell lysate after a 30
min. .sup.35S-Met-Cys pulse-labelling. The expression pattern is
essentially identical to that observed in E. coli (see FIG. 4). The
DNA transfected into the cells (or cotransfected) is indicated
above the respective lanes.
FIG. 7 illustrates the results of a protein engineering application
of the mDHFR bacterial PCA. Two semi-random leucine zipper
libraries were created (as described in the text) and each inserted
N-terminal to one of the mDHFR fragments. Cotransformation of the
resulting zipper-DHFR fragment libraries in E. coli and plating on
selective medium allowed for survival of clones harboring
successfully interacting leucine zippers. Fourteen clones were
isolated and the zippers were sequenced to identify the residues at
the "e" and "g" positions. The "e-g" pairs were categorized, as
having attractive pairing (charge:charge, charge:neutral polar or
neutral polar:neutral polar) or repulsive pairing (charge:charge)
and the number of each type of interaction scored for each clone.
The total number of interactions for each clone is 6; the
interactions are tallied on the histogram.
Other objects, advantages and features of the present invention
will become more apparent upon reading of the following
non-restrictive description of preferred embodiments with reference
to the accompanying drawings which are exemplary and should not be
interpreted as limiting the scope of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Selection of mDHFR for a PCA
In designing a protein-fragment complementation assay (PCA), we
sought to identify an enzyme for which the following is true: 1) An
enzyme that is relatively small and monomeric, 2) for which
structural and functional information exists, 3) for which simple
assays exist for both in vivo and in vitro measurement, and 4) for
which overexpression in eukaryotic and prokaryotic cells has been
demonstrated. Murine DHFR (mDHFR) meets all of the criteria for a
PCA listed above. Prokaryotic and eukaryotic DHFR is central to
cellular one-carbon metabolism and is absolutely required for cell
survival in both prokaryotes and eukaryotes. Specifically it
catalyses the reduction of dihydrofolate to tetrahydrofolate for
use in transfer of one-carbon units required for biosynthesis of
serine, methionine, purines and thymidylate. The DHFRs are small
(17 kD to 21 kD), monomeric proteins. The crystal structures of
DHFR from various bacterial and eukaryotic sources are known and
substrate binding sites and active site residues have been
determined.sup.111-114, allowing for rational design of protein
fragments. The folding, catalysis, and kinetics of a number of
DHFRs have been studied extensively.sup.115-119. The enzyme
activity can be monitored in vitro by a simple spectrophotometric
assay.sup.120, or in vivo by cell survival in cells grown in the
absence of DHFR end products. DHFR is specifically inhibited by the
anti-folate drug trimethoprim. As mammalian DHFR has a 12000-fold
lower affinity for trimethoprim than does bacterial DHFR.sup.121,
growth of bacteria expressing mDHFR in the presence of trimethoprim
levels lethal to bacteria is an efficient means of selecting for
reassembly of mDHFR fragments into active enzyme. High level
expression of mDHFR has been demonstrated in transformed prokaryote
or transfected eukaryotic cells.sup.122-126.
Design Considerations.
mDHFR shares high sequence identity with the human DHFR (hDHFR)
sequence (91% identity) and is highly homologous to the E. coli
enzyme (29% identity, 68% homology) and these sequences share
visually superimposable tertiary structure.sup.111. Comparison of
the crystal structures of mDHFR and hDHFR suggests that their
active sites are essentially identical.sup.127,128. DHFR has been
described as being formed of three structural fragments forming two
domains.sup.129, 130 the adenine binding domain (residues 47 to
105=fragment[2]) and a discontinuous domain (residues 1 to
46=fragment[1] and 106 to 186 [3]; numbering according to the
murine sequence). The folate binding pocket and the NADPH binding
groove are formed mainly by residues belonging to fragments[1] and
[2]. Fragment [3] is not directly implicated in catalysis.
Residues 101 to 108 of hDHFR, at the junction between fragment[2]
and fragment[3], form a disordered loop which lies on the same face
of the protein as both termini. We chose to cleave mDHFR between
fragments [1,2] and [3], at residue 107, so as to cause minimal
disruption of the active site and NADPH cofactor binding sites. The
native N-terminus of mDHFR and the novel N-terminus created by
cleavage occur on the same surface of the enzyme.sup.112, 128
allowing for ease of N-terminal covalent attachment of each
fragment to associating fragments such as the leucine zippers used
in this study. Using this system, we have obtained leucine-zipper
assisted assembly of the mDHFR fragments into active enzyme.
Example 1
Experimental Protocol
DNA Constructs.
Mutagenic and sequencing oligonucleotides were purchased from Gibco
BRL. Restriction endonucleases and DNA modifying enzymes were from
Pharmacia and New England Biolabs. The mDHFR fragments carrying
their own iN-frame stop codon were subcloned into pQE-32 (Qiagen),
downstream from and iN-frame with the hexahistidine peptide and a
GCN4 leucine zipper (FIG. 1; FIG. 2). All final constructs were
based on the Qiagen pQE series of vectors, which contain an
inducible promoter-operator element (tac), a consensus ribosomal
binding site, initiator codon and nucleotides coding for a
hexahistidine peptide. Full-length mDHFR is expressed from pQE-16
(Qiagen).
Expression Vector Harboring the GCN4 Leucine Zipper
Residues 235 to 281 of the GCN4 leucine zipper (a SalI/BamHI 254 bp
fragment) were obtained from a yeast expression plasmid
pRS316.sup.9. The recessed terminus at the BamHI site was filled-in
with Klenow polymerase and the fragment was ligated to pQE-32
linearized with SalI/HindIII (filled-in). The product, construct Z,
carries an open reading frame coding for the sequence
Met-Arg-Gly-Ser followed by a hexahistidine tag and 13 residues
preceding the GCN4 leucine zipper residues.
Creation of DHFR Fragments:
The eukaryotic transient expression vector, pMT3 (derived from
pMT2).sup.16, was used as a template for PCR-generation of mDHFR
containing the features allowing subcloning and separate expression
of fragment[1,2] and fragment[3]. The megaprimer method of PCR
mutagenesis.sup.29 was used to generate a full-length 590 bp
product. Oligonucleotides complementary to the nucleotide sequence
coding for the N- and C-termini of mDHFR and containing a novel
BspEI site outside the coding sequence were used as well as an
oligonucleotide used to create a novel stop codon after
fragment[1,2], followed by a novel SpeI site for use in subcloning
fragment[3].
Construction of a New Multiple Cloning Region and Subcloning of
DHFR Fragments [1,2] and [3]
Complementary oligonucleotides containing the novel restriction
sites: SnaBI, NheI, SpeI and BspEI, were hybridized together
resulting in 5' and 3' overhangs complementary to EcoRI, and
inserted into pMT3 at a unique EcoRI site. The 590 bp PCR product
(described above) was digested with BspEI and inserted into pMT3
linearized at BspEI, yielding construct [1,2,3]. The 610 bp
BspEI/EcoNI fragment (coding for DHFR fragment[1,2], followed by a
novel stop and fragment[3] up to EcoNI) was filled in at EcoNI and
subcloned into pMT3 opened with BspEI/HpaI, yielding construct
F[1,2]. The 250 bp SpeI/BspEI fragment of construct [1,2,3] coding
for DHFR fragment[3] (with no in-frame stop codon) was subcloned
into pMT3 opened with the same enzymes. The stop codon of the
wild-type DHFR sequence, downstream from fragment[3] in pMT3, was
inserted as follows. Cleavage with EcoNI, present in both the
inserted fragment[3] and the wild-type fragment[3], removal of the
683 bp intervening sequence and relegation of the vector yielded a
construct of fragment[3] with the wild-type stop codon, construct
F[3].
Creation of the Expression Constructs
The 1051 bp and the 958 bp SnaBI/XbaI fragments of constructs
F[1,2] and F[3], respectively, were subcloned into construct Z
opened with BglII (filled-in)/NheI, yielding constructs Z-F[1,2]
and Z-F[3] (FIG. 2). For the Control expression construct, the 180
bp XmaI/BspEI fragment coding for the zipper was removed from
construct Z-F[1,2], yielding construct Control-F[1,2] (FIG. 2).
Creation of Stability Mutants
Site-directed mutagenesis was performed.sup.30 to produce mutants
at Ile114 (numbering of the wild-type mDHFR). The mutagenesis
reaction was carried out on the KpnI/BamHI fragment of construct
Z-F[3] subcloned into pBluescript SK+ (Stratagene), using
oligonucleotides that encode a silent mutation producing a novel
BamHI site. The 206 bp NheI/EcoNI fragment of putative mutants
identified by restriction was subcloned back into Z-F[3]. The
mutations were confirmed by DNA sequencing.
E. coli Survival Assay
E. coli strain BL21 carrying plasmid pRep4 (from Qiagen, for
constitutive expression of the lac repressor) were made competent,
transformed with the appropriate DNA constructs and washed twice
with minimal medium before plating on minimal medium plates
containing 50 mg/ml kanamycin, 100 mg/ml ampicillin and 0.5 mg/ml
trimethoprim. One half of each transformation mixture was plated in
the absence, and the second half in the presence, of 1 mM IPTG. All
plates were placed at 37.degree. C. for 66 hrs.
E. coli Growth Curves
Colonies obtained from cotransformation were propagated and used to
inoculate 10 ml of minimal medium supplemented with ampicillin,
kanamycin as well as IPTG (1 mM) and trimethoprim (1 .mu.g/.mu.l)
where indicated. Cotransformants of Z-F[1,2]+Z-F[3:Ile114Gly] were
obtained under non-selective conditions by plating the
transformation mixture on L-agar (+kanamycin and ampicillin) and
screening for the presence of the two constructs by restriction
analysis. All growth curves were performed in triplicate. Aliquots
were withdrawn periodically for measurement of optical density.
Doubling time was calculated for early logarithmic growth (OD 600
between 0.02 and 0.2).
Protein Overexpression and Purification
Bacteria were propagated in Terrific Broth.sup.31 in the presence
of the appropriate antibiotics to an OD600 of approximately 1.0.
Expression was induced by addition of 1 mM IPTG and further
incubation for 3 hrs. For analysis of crude extract, pellets from
150 ml of induced cells were lysed by boiling in loading dye. The
lysates were clarified by microcentrifugation and analyzed by
SDS-PAGE32. For protein purification, a cell pellet from 50 ml of
induced E. coli cotransformed with constructs Z-F[1,2] and Z-F[3]
was lysed by sonication, and a denaturing purification of the
insoluble pellet undertaken using Ni-NTA (Qiagen) as described by
the manufacturer. The proteins were eluted with a stepwise
imidazole gradient. The fractions were analyzed by SDS-PAGE.
Results
Design of mDHFR Fragments for a PCA
mDHFR shares high sequence identity with the human DHFR (hDHFR)
sequence. As the coordinates of the murine crystal structure were
not available, we based our design considerations on the hDHFR
structure. DHFR has been described as comprising three structural
fragments forming two domains: the adenine binding domain (F[2])
and a discontinuous domain (F[1] and F[3]).sup.13,18. The folate
binding pocket and the NADPH binding groove are formed mainly by
residues belonging to F[1] and F[2]. Residues 101 to 108 of hDHFR
form a disordered loop which lies on the same face of the protein
as both termini. This loop occurs at the junction between F[2] and
F[3]. By cleaving mDHFR at residue 107, we created F[1,2] and F[3],
thus causing minimal disruption of the active site and substrate
binding sites. The native N-terminus of mDHFR and the novel
N-terminus created by cleavage were covalently attached to the
C-termini of GCN4 leucine zippers (FIG. 1).
E. coli Survival Assays
FIG. 2 illustrates the general features of the expressed constructs
and the nomenclature used in this study. FIG. 3 (panel A)
illustrates the results of cotransformation of bacteria with
constructs coding for Z-F[1,2] and Z-F[3], in the presence of
trimethoprim, clearly showing that colony growth under selective
pressure is possible only in cells expressing both fragments of
mDHFR. There is no growth in the presence of either Z-F[1,2] or
Z-F[3] alone. Induction of protein expression with IPTG is
essential for colony growth (FIG. 3A). The presence of the leucine
zipper on both fragments of mDHFR is essential as illustrated by
cotransformation of bacteria with both vectors coding for mDHFR
fragments, only one of which carries a leucine zipper (FIG. 3A). It
should be noted that growth of control E. coli transformed with the
full-length mDHFR is possible in the absence of IPTG due to low
levels of expression in uninduced cells.
Confirmation of the presence of both plasmids in bacteria able to
grow with trimethoprim was obtained from restriction analysis of
the plasmid DNA purified from isolated colonies. FIG. 4 (A) reveals
the presence of the 1200 bp HincII restriction fragment from
construct Z-F[1,2] as well as the 487 and 599 bp HincII restriction
fragments from construct Z-F[3]. Also present is the 935 bp HincII
fragment of pRep4. Overexpression of the fusion proteins is
illustrated in FIG. 4 (B). In all cases, overexpression of a
protein of the expected molecular weight is apparent on SDS-PAGE of
the crude lysate. Purification of the coexpressed proteins under
denaturing conditions yielded two bands of apparent homogeneity
upon analysis by Coomassie-stained SDS-PAGE (FIG. 4B).
Stability Mutants
Applicants generated mutants of F[3] to test whether reconstitution
of mDHFR activity by fragment assembly was specific. Protein
stability can be reduced by changing the side-chain volume in the
hydrophobic core of a protein.sup.9, 22-25. Residue Ile114 of mDHFR
occurs in a core .beta.-strand at the interface between F[1,2] and
F[3], isolated from the active site. Ile 114 is in van der Waals
contact with Ile51 and Leu93 in F[1,2].sup.11. We mutated Ile 114
to Val, Ala, or Gly. FIG. 3 (panel B) illustrates the results of
cotransformation of E. coli with construct Z-F[1,2] and the mutated
Z-F[3] constructs. The colonies obtained from cotransformation with
Z-F[3:Ile114Ala] grew more slowly than those cotransformed with
Z-F[3] or Z-F[3:Ile114Val] (see inset to FIG. 3B). No colony growth
was detected in cells cotransformed with Z-F[3:Ile114Gly]. The
number of transformants obtained was not significantly different in
the case where colonies were observed, implying that cells
cotransformed with Z-F[1,2] and either Z-F[3], Z-F[3:Ile114Val] or
Z-F[3:Ile114Ala] have an equal survival rate. Overexpression of the
mutants Z-F[3:Ile114X] was in the same range as Z-F[3], as
determined by Coomassie-stained SDS-PAGE (data not shown).
We also compared the relative efficiency of reassembly of mDHFR
fragments by measuring the doubling time of the cotransformants in
liquid medium. Doubling time in minimal medium was constant for all
transformants (data not shown). Selective pressure by trimethoprim
in the absence of IPTG prevented growth of E. coli except when
transformed with pQE-16 coding for full-length DHFR due to low
levels of expression in uninduced cells. Induction of mDHFR
fragment expression with IPTG allowed survival of cotransformed
cells (except in the case of Z-F[1,2]+Z-F[3:Ile114Gly], although
the doubling times were significantly increased relative to growth
in the absence of trimethoprim. The doubling time measured for
cells expressing Z-F[1,2]+Z-F[3], Z-F[1,2]+Z-F[3:Ile114Val] and
Z-F[1,2]+Z-F[3:Ile114Ala] were 1.6-fold, 1.9-fold and 4.1-fold,
higher respectively, than the doubling time of E. coli expressing
pQE-16 in the absence of trimethoprim and IPTG. The presence of
IPTG unexpectedly prevented growth of E. coli transformed with
full-length mDHFR. Growth was partially restored by addition of the
folate metabolism end-products thymine, adenine, pantothenate,
glycine and methionine (data not shown). This suggests that induced
overexpression of mDHFR was lethal to E. coli when grown in minimal
medium as a result of depletion of the folate pool by binding to
the enzyme.
In another embodiment, applicants make point mutations in the GCN4
leucine zipper of Z-F[1,2] and Z-F[3], for which direct equilibrium
and kinetic parameters are known and correlating these known values
with parameters derived from the PCA (Pelletier and Michnick, in
preparation). Comparison of cell growth rates in this model system
with rates for a DHFR PCA using unknowns would give an estimate of
the strength of the unknown interaction. This should enable the
determination of estimates of equilibrium and kinetic parameters
for a specific protein-protein interaction.
The present invention has illustrated and demonstrated a
protein-fragment complementation assay (PCA) based on mDHFR, where
a leucine zipper directs the reconstitution of DHFR activity.
Activity was detected by an E. coli survival assay which is both
practical and inexpensive. This system illustrates the use of mDHFR
fragment complementation in the detection of leucine zipper
dimerization and could be applied to the detection of unknown,
specific protein-protein interactions in vivo.
E. coli Aminoglycoside Kinase: Optimization and Design of a PCA
Using an Exonuclease-Molecular Evolution Strategy
Although applicants have demonstrated that the engineering/design
strategy described above can be used to produce complementary
enzyme fragments, it is obvious that proteins did not evolve in
such a way that such fragments would be expected to have optimal
physical characteristics, including solubility, foldability (fast
folding), protease resistance, or enzymatic activity. An
alternative embodiment to the engineering/design strategy is the
endonuclease/evolution approach. This strategy can be used by
itself or in conjunction with the engineering/design strategy. The
advantages of this approach are that in principle, prior knowledge
of the protein structure is not necessary, that the optimal
fragments are chosen for PCA and that these fragments will also
have optimal characteristics. Following selection of optimal
complementary fragments, the fragments are exposed to multiple
rounds of random mutagenesis. Mutagenesis is achieved by suboptimal
PCR combined with chemical mutagenesis or DNA shuffling (Stemmer,
W. P. C. (1994) Proc, Natl, Acad, Sci. USA 91, 10747-10751). The
overall strategy is described for the case of aminoglycoside kinase
(AK), an example of antibiotic resistance marker that can be used
for dominant selection of prokaryotic cells such as E. coli or
eukaryotic cells such as yeast or mammalian cell lines. The
structure of an AK is already known, and so strategy (1) would be
possible, however we chose to combine both strategy (1) as defined
for DHFR above, in conjunction with strategy (2).
Experimental Protocol
The optimization/selection procedure is as follows:
Generation of Library of AK Fragments Based on Products of
Exonuclease Digestion
Nested sets of deletions are created at the 5' and the 3' ends of
the AK gene. In order to create unidirectional deletions, unique
restriction sites are introduced in the regions flanking the AK
gene. At the 5' and 3' termini, an "outer" sticky site with a
protruding 3' terminus (Sph I and Kpn I, respectively) and an
"inner" sticky site with recessed 3' terminus (Bgl II and Sal I,
respectively) are added by PCR. Cleavage at Sph I and Bgl II (or
Kpn I and Sal I) results in creation of a protruding terminus
leading back to the flanking sequence and a recessed terminus
leading into the AK gene. Digestion with E. coli exonuclease III
and S1 nuclease (Henikoff, S. (1987) Methods in Enzymology 155,
156-165) yields a set of nested deletions from the recessed
terminus only. Thus, 10 mg of DNA is digested with Sph I and Bgl II
(or Kpn I and Sal I), phenol-chloroform extracted, and 12.5 U
exonuclease III added. At 30 sec intervals over 10 min, aliquots
are taken and put into solution with 2 U S1 nuclease. The newly
created ends are filled in with T4 DNA polymerase (0.1 Upper
sample) and the set of vectors closed back by blunt-ended ligation
(10 U ligase per sample). The average length of the deletion at
each time point is determined by restriction analysis of the sets.
This yields sets of AK genes deleted from the 5' or the 3' termini.
This manipulation is undertaken directly in the pQE-32-Zipper
constructs, such that the products can be used directly in activity
screening.
Screening for AK Activity
As a first step in determining the requirements for fragment
complementation, we must determine the minimum N-terminal and
C-terminal fragments of AK that, alone, are active. Sets of
deletions are individually transformed into E. coli BL21 cells and
expression of the AK fragments is induced by IPTG. The sets where a
significant number of colonies appear in the presence of G418 serve
to indicate the approximate length of N- and C-terminal AK
fragments which retain activity. Fragment complementation must
therefore be undertaken with fragments taken from within these
limits. The zipper-directed fragment complementation is detected as
follows: appropriate sets of deletions, or pools of sets, are
cotransformed into BL21, expression is induced with IPTG and growth
in the presence of varying G418 concentrations is monitored. Large
colonies which grow in the presence of high G418 concentrations are
selected as giving the most efficiently complementing products.
Directed evolution of optimal AK fragments using "DNA shuffling
After optimal fragments have been selected, the individual
fragments are removed by restriction digestion at Sph I and Kpn I
allowing for 5' and 3' constant priming regions flanking the N- or
C-terminal complementary fragments of AK. These oligonucleotides
(2-4 .mu.g) are digested with DNaseI (0.005 units/ul, 100 ul) and
fragments of 10-50 nucleotides are extracted from low melting point
agarose. PCR is then performed with the fragmented DNA, using Taq
polymerase (2.5 units/up in a PCR mixture containing 0.2 mM dNTPs,
2.2 mM Mg2Cl (or 0 mM for subuptimal PCR), 50 mM KCl, 10 mM
Tris.HCl, pH 9.0, 0.1% TritonX-100. A PCR program of 94 C/60 sec.;
94 C 30 sec.; 55 C 30 sec.; 72 C 30 sec. times 30 to 50; 72 C 5
min. Samples are taken every 5 cycles after 25 cycles to monitor
the appearance of reassembled complete fragments on agarose gel.
The primerless PCR product is then diluted 1:40 or 1:60 and used as
template for PCR with 5', 3' complementary constant region oligos
as primers for a further 20 cycles. Final product is restriction
digested with Sph I and Kpn I and the products subcloned back into
pQE32-Zipper to yield the final library of expression plasmids. As
before, E. coli BL21 cells are sequentially transformed with
C-terminal or N-terminal complementary fragment-expression vectors
at an estimated efficiency of 109 and finally cells cotransformed
with the complementary fragment. E. coli are grown on agarose
plates containing 1 .mu.g/ml G418 and after 16 hours the largest
colonies are selected and grown in liquid medium at increasing
concentrations of G418. Those clones showing the maximal resistance
to G418 are then selected and if maximum resistance or greater is
reached the evolution is terminated. Otherwise the DNA shuffling
procedure is repeated. Finally, optimal fragments are sequenced and
physical properties and enzymatic activity are assessed. This
optimized AK PCA is now ready to test for dominant selection in any
other cell type including yeast and mammalian cell lines. This
strategy can be used to develop any PCA based on enzymes that
impart dominant or recessive selection to a drug or toxin or to
enzymes that produce a colored or fluorescent product. In the later
two cases the end point of the evolution process is at minimum,
reatainment of signal for the intact, wild type enzyme or
enhancement of the signal. This strategy can also be used in the
absence of knowledge of the enzyme structure, whether the enzyme in
mono-, di- or multimeric structure. However, knowledge of the
enzyme structure does not preclude applying this strategy as well,
as described below.
As can be appreciated, knowledge of the enzyme structure can be
used to render a more efficient way of using molecular evolution to
design a PCA. In this case, the enzyme structure is used to define
minimal domains of the protein in question, as was done for DHFR.
Instead of generating fragments of completely random length for the
N- or C-terminal fragments, we select, during the exonuclease
phase, those fragments that at a minimum will code for one of the
two domains. For instance, in the case of AK, two well defined
domains can be discerned in the structure consisting of residues
1-94 in the N-terminus and residues 95-267 in the C-terminus.
Endonuclease digestions are performed as above, but reaction
products are selected that will minimally code for one of the two
domains. These are then the starting points for fragment selection
and evolution cycles as described above.
Heteromeric Enzyme PCA
A further embodiment of the invention relates to PCA based on using
heterodimeric or heteromultimeric enzymes in which the entire
catalytic machinery is contained within one independently folding
subunit and the other subunit provides stability and/or a cofactor
to the enzymatic subunit. In this embodiment of PCA, the regulatory
subunit is split into complementary fragments and fused to
interacting proteins. These fragments are
co-transformed/transfected into cells along with the enzyme
subunit. As with single enzyme PCA described for DHFR and AK,
reconstitution and detection of enzyme activity is dependent on
oligomerization domaiN-assisted reassembly of the regulatory
subunit reassembly into its native topology. However, the
reconstituted subunit then interacts with the intact enzymatic
subunit to produce activity. This approach is reminiscent of the
USPS system, except it has the advantage that the enzyme in this
case is not a constitutive cellular enzyme, but rather an exogenous
gene product. As such there is no problem with background activity
from the host cell, the enzyme can be expressed at higher levels
than a natural gene and can also be modified to be directed to
specific subcellular compartments (by subcloning
compartment-specific signal peptides onto the N- or C-termini of
the enzyme and subunit fragments). The specific advantage of this
approach is that while the single enzyme strategy may lead to
suboptimal enzymatic activity, in this approach, the enzyme folds
independently and may in fact act as a chaperone to the fragmented
regulatory subunit, aiding in its refolding. In addition, folding
of the fragments may need not be complete in order to impart
regulation of the enzyme. This approach is realized by a
colorimetric/fluorometric assay we have developed based on the
Streptomyces tyrosinase. This enzyme catalyzes the conversion of
tyrosine to deoxyphenylalanine (DOPA). The reaction can be measured
by conversion of fluorocinyl-tyrosine to the DOPA form. The active
enzyme consists of two subunits, the catalytic domain (Melc2) and a
copper binding domain (Melc1). Meld is a small protein of 14 kD
that is absolutely required for Melc2 activity. In the assay we are
developing, the Meld protein is split into two fragments that serve
as the complementation part of the PCA. These fragments, fused to
oligomerization domains, are coexpressed with Melc2, and the basis
of the assay is that Melc2 activity is dependent on complementation
of the Meld fragments. Stoichiometries of protein complexes can
also be addressed (i.e. whether a complex consists of two or three
proteins) as follows. One fuses two proteins to the two Melc1
fragments and a third to intact Melc2. It thus can be shown that
the minimum complementary active complex of the tyrosinase will
require that all three components and therefore a trimer is
necessary. A key aspect of this approach is that we can easily
demonstrate specific interactions by making one component,
specifically the protein-Melc2 fusions catalytic subunit dependent
on the other components by underexpressing it in the background of
overexpressed Melc1 fragment-protein fusions.
Multimer Disruption-Based PCA
Although applicants have described only fragment complementation of
intact proteins, protein domains or subunits as comprising PCA, an
alternate embodiments relates to PCAs based on the disruption of
the interface between, for instance a dimeric enzyme that requires
stable association of the subunits for catalytic activity. In such
cases, selective or random mutagenesis at the subunit interface
would disrupt the interaction and the basis of the assay would be
that oligomerization domains fused to the subunits would provide
the necessary binding energy to bring the subunits together into a
functional enzyme. Vector Design in Application to PCAs
The PCA strategies listed thus far have used two-plasmid
transformation strategies for expression of complementary
fragments. This approach has some advantages, such as using
different drug resistance markers to select for optimal
incorporation of genes, for instance in transformed or transfected
cells or for optimum transformation of complementary plasmids into
bacteria and control of expression levels of PCA fragments using
different promoters. However, single plasmid strategies have
advantages in terms of simplicity of transfection/transformation.
Protein expression levels can be controlled in different ways,
while drug selection can be achieved in one of two ways: In the
case of PCAs based on survival assay using enzymes that are drug
resistance markers themselves, such as AK, or where the enzyme
complements a metabolic pathway, such as DHFR, no additional drug
resistance genes need be incorporated in the expression plasmids.
If however the PCA is based on an enzyme that produces a colored or
fluorescent product, such as tyrosinase or firefly luciferase, an
additional drug resistance gene must be expressed from the plasmid.
Expression of PCA complementary fragments and fused cDNA
libraries/target genes can be assembled on single plasmids as
individual operons under the control of separate inducible or
constitutive promoters, or can be expressed polycistronically. In
E. coli polycistronic expression can be achieved using known
intercoding region sequences, for instance we use the region in the
mel operon from which we derived the tyrosinase melc1-melc2 genes
which we have shown to be expressed at high levels in E. coli under
the control of a strong (tac) promoter. Genes could also be
expressed and induced off of independent promoters, such as tac and
arabinose. For mammalian expression systems, single plasmid systems
can be used for both transient or stable cell line expression and
for constitutive or inducible expression. Further, differential
control of the expression of one of the complementary fragment
fusions, usually the bait-fused fragment, can be controlled to
minimize expression. This will be important in reducing background
non-specific interactions. Examples of differential control of
complementary fragment expression include the following
strategies:
i) In polycistronic expression, transient or stable, expression of
the second gene will necessarily be less efficient and so this in
itself could serve to limit the quantity of one of the
complementary fragments. Alternatively, the first gene product can
be limited in expression by mutation of an upstream donor/splice
site, while the second gene can be put under the control of a
retroviral internal initiation site, such as that of ECMV to
enhance expression. ii) Individual complementary fragment-fusion
pairs can also be put under the control of inducible promoters, all
commercially available including those based on Tet-responsive
PhCMV*-1 promoter, and/or steroid receptor response elements. In
such a system the two complementary fragment genes can be turned on
and expression levels controlled by dose dependent expression with
the inducer, in these cases tetracycline and steroid hormones.
Example 2
Applications of the PCA Strategy to Detect Novel Gene Products in
Biochemical Pathways and to Map Such Pathways
Among the greatest advantage of PCA over other molecular
interaction screening methods is that they are designed to be
performed both in vivo and in any type of cell. This feature is
crucial if the goal of applying a technique is to identify novel
interactions from libraries and simultaneously be able to determine
if the interactions observed are biologically relevant. The
detailed example given below, and other examples at the end of this
section illustrate how it is that validation of interactions with
PCA is possible. In essence, this is achieved as follows. In
biochemical pathways, such as hormone receptor-mediated signaling,
a cascade of enzyme-mediated chemical reactions are triggered by
some molecular event, such as by hormone binding to its membrane
surface receptor. Enzyme interactions with protein substrates and
protein-protein or protein-nucleic acid interactions with
enzyme-modified substrates then occur. Such biochemical signaling
cascades only occur in specific cell types and model cell lines for
studying these processes. Therefore, to detect induced
interactions, such as with known proteins in a pathway with yet
unidentified proteins, one obviously needs to perform such
screening in appropriate model cell lines and in the correct
cellular compartment. Only the PCA strategy can be used in a
general way to do this. Protein-molecular interaction techniques
such as yeast two- or three-hybrid techniques cannot be performed
in a context where such events occur, except in the limiting case
of nuclear interaction in yeast or interactions that are not
triggered. There do exist mammalian two-hybrid techniques where it
might be possible to detect induced protein interactions, but only
again if the proteins involved can be simultaneously activated,
transported to the nucleus and interact with their partners. PCAs
do not have these limitation since they do not require additional
cellular machinery available only in specific compartments. A
further point is that by performing the PCA strategy in appropriate
model cell types, it is also possible to introduce appropriate
positive and negative controls for studying a particular pathway.
For instance, for a hormone signaling pathway it is likely that
hormone signaling agonists and antagonists or dominant-negative
mutants of signaling cascade proteins would be known, that are
upstream or act in parallel to the events being examined in the
PCA. These reagents could be used to determine if novel
interactions detected by the PCA are biologically relevant. In
general then, interactions that are detected only if hormone is
introduced but are not seen if an antagonist is simultaneously
introduced could be hypothesized to represent interactions relevant
to the process under study.
Below is a detailed description of an application of the DHFR that
illustrates these points, as well as further examples where the PCA
strategy could be used.
Application of the DHFR PCA to Mapping Growth Factor-Mediated
Signal Transduction Pathways
One of the earliest detectable events in growth factor-activated
cell proliferation is the serine phosphorylation of the S6 protein
of the 40S ribosomal subunit. The discovery of serine/threonine
kinases that specifically phosphorylate S6 have considerably aided
in identifying novel mitogen mediated signal transduction pathways.
The serine/threonine kinase p70S6k has been identified as a
specific S6 phosphorylase.sup.131-136. p70S6k is activated by
serine and threonine phosphorylation at specific sites in response
to several mitogenic signals including serum in serum starved
cells, growth factors including insulin and PDGF, and by mitogens
such as phorbol esters. Considerable effort has been made over the
last five years to determine how p70/p85S6k are activated in
response to mitogens. Two receptor-mediated pathways have been
implicated in p70S6k activation, one associated with the
phosphatidylinositol-3-kinase (PI(3)k) and the other with the
PI(3)k homologue mTOR.sup.137-144. Key to understanding of this
proposal, is the fact that the role of these enzymes in activation
of p70S6k was determined by effects of two natural products on
phosphorylation and enzyme activity: rapamycin, which indirectly
inhibits mTOR activity, and wortmannin, which directly inhibits
PI(3).sub.k activity. It is also important to note that no direct
upstream kinases or other regulatory proteins of p70S6k have been
identified to this date.
The interactions of p70S6k with its known substrate S6 can be
studied as a test system for the DHFR PCA in E. coli and in
mammalian cell lines. One can also seek to identify novel
interactions with this enzyme that would lead to new insights into
how this important enzyme is regulated. Also, since activation of
the enzyme is mediated by multiple pathways that can be selectively
inhibited with specific drugs, this is an ideal system to test PCAs
as methods to distinguish induced versus constitutive
protein-protein interactions.
a) Testing of the E. coli Survival Assay: Interaction of p70S6k
with S6
This test is ideal, because the apparent Km (=250 nM) of p70S6k for
S6 protein.sup.145 is approximately the same as the Kd for leucine
zipper-forming peptides from GCN4 used in our test system. However,
we will have to use a constitutively active form of the enzyme for
our tests. An N-terminal truncated form of the enzyme D77-p70S6k,
is constitutively active and will be used in these studies147.
Methodology: D77-p70S6k-F[1,2] fusion and D77-p70S6k-F[3] fusion,
or F[1,2] and D77-p70S6k-F[3] fusion (as a control) will be
cotransformed into E. coli and the cells grown in minimal medium in
the presence of trimethoprim. Colonies will be selected and
expanded for analysis of kinase activity against 40S ribosomal
subunits, and for coexpression of the two proteins. b) Modification
of the Bacterial Survival Assay for Library Screening:
Identification of Novel Interacting Proteins.
Screening an expression library for interactions with a given
target (p70S6k-D77, in this case) will be straightforward in this
system, given that the only steps involved are: 1-construction of
the fusion-expression library as a fusion with mDHFR fragment[3];
2-transformation of the library in E. coli BL21 harboring pRep4
(for constitutive expression of the lac repressor; this is required
in the case where a protein product is toxic to the cells) and a
plasmid coding for the fusion: p70S6k-D77-[1,2]; 3-plating on
minimal medium in the presence of trimethoprim and IPTG;
4-selection of any colonies that grow, propagation and isolation of
plasmid DNA, followed by sequencing of DNA inserts; 5-purification
of unknown fusion products via the hexaHis-tag and sizing on
SDS-PAGE.
Methodology:
The overall strategy is illustrated in FIG. 5. 1-Construction of a
directional fusioN-expression library: i-cDNA production: One can
isolate poly(A)+ RNA from BA/F3 cells (B-lymphoid cells) because
these cells have successfully been used in the study of the
rapamycin-sensitive p70S6k activation cascade.sup.139. To enrich
for full-length mRNA, we will affinity purify the mRNA via the 5'
cap structure by the CAPture method.sup.148. Reverse transcription
will be primed by a "Linker Primer": it has a poly(T) tail to prime
from the poly(A) mRNA tail, and an XhoI site for later use in
directional subcloning of the fragments. The first strand is then
methylated. After second strand synthesis and blunting of the
products, "EcoRI Adapters" are added, producing digestion of the
linkers with EcoRI and XhoI (the inserts are protected by
methylation) produces full-length cDNA ready for directional
insertion in a vector opened with EcoRI and XhoI. Because the
success of library screening depends largely on the quality of the
cDNA produced, we will use the above methods as they have proven to
consistently produce high-quality cDNA libraries. ii-Insertion of
the cDNA into vectors: The library will be constructed as a
C-terminal fusion to mDHFR F[3] in vector pQE-32 (Qiagen), as we
have obtained high levels of expression of mDHFR fusions from this
vector in BL21 cells. Three such vectors will be created, differing
at their 3' end, which is the novel polycloning site that we
engineered (described earlier, under Methods), carrying either 0,
1, or 2 additional nucleotides. This allows read-through from F[3]
into the library fragments in all 3 translational reading frames.
The cDNA fragments will be directionally inserted at the EcoRI and
XhoI sites in all three vectors at once. 2, 3, 4, and 5--These
steps have been described earlier, under Results, apart from the
final sequencing of clones identified using sequencing primers
specific to vector sequences flanking sites of library insertion.
The protein purification will also be as described earlier, by a
one-step purification on Ni-NTA (Qiagen). If the product size is
more than 15 kDa over the molecular weight of the DHFR component
(equal to a cDNA insert of more than 450 bp), we will have the
inserts sequenced at the Sheldon Biotechnology Center (McGill
University).
c) Development of the Eukaryotic Assay
The transformation of the system described above, is useful to
produce an equivalent assay for use in eukaryotic cells. The basic
principle of the assay is the same: the fragments of mDHFR are
fused to associating domains, and domain association is detected by
reconstitution of DHFR activity in eukaryotic cells (FIG. 5).
Creation of the expression constructs: The DNA fragments coding for
the GCN4-zipper-mDHFR fragment fusions were inserted as one piece
into pMT3, a eukaryotic transient expression vector.sup.126.
Expression of the fusion proteins in COS cells was apparent on
SDS-PAGE after 35[S]Met labeling. Survival assays in eukaryotic
cells: Two systems can be used for detection of mDHFR reassembly,
in parallel: i--CHO-DUKX B11 cells (Chinese Hamster Ovary cell line
deficient in DHFR activity) are cotransfected with
GCN4-zipper-mDHFR fragment fusions. The cells are grown in the
absence of nucleotides; only cells carrying reconstituted DHFR will
undergo normal cell division and colony formation. ii-Methotrexate
(MTX)-resistant mutants of mDHFR have been created, with the goal
of transfecting cells that have constitutive DHFR activity such as
COS and 293 cells. We mutated F[1,2] in order to incorporate, one
at a time, each of five mutations that significantly increase Ki
(MTX): Gly15Trp, Leu22Phe, Leu22Arg, Phe31Ser and Phe34Ser
(numbering according to the wild-type mDHFR sequence). These
mutations occur at varying positions relative to the active site
and relative to F[3], and have varying effects on Km (DHF), Km
(NADPH) and Vmax of the full-length mammalian enzymes in which they
were. Mutants Z-F[1,2: Leu22Phe], Z-F[1,2: Leu22Arg] and Z-F[1,2:
Phe31Ser] all allowed for bacterial survival with high growth rates
when cotransformed with Z-F[3] (results not shown). The five
mutants will be tested in eukaryotic cells, in reconstitution of
mDHFR fragments to produce enzyme that can sustain COS or 293 cell
growth while under the selective pressure of MTX, which will
eliminate background due to activity of the native enzyme. The
mutations offers an advantage in selection while presenting no
apparent disadvantage with respect to reassembly of active enzyme.
If the reconstituted mDHFR produced in either of the survival
assays allows eukaryotic cell growth that is significantly slower
than growth with the wild-type enzyme, thymidylate will be added to
the growth medium to partially relieve the selective pressure
offered by the lack of nucleotides. d) Testing of the Eukaryotic
Survival Assay
It is necessary at the outset to test whether induced interactions
with p70S6k can be detected. One can use the same test system as
that for the E. coli test system described above: Induction of
association of p70S6k with S6 protein.
Methodology:
mDHFR Leu22Phe mutant S6-F[1,2] and p70S6k-F[3], or F[1,2] and
p70S6k-F[3] (as a control) will be cotransfected into COS cells and
the cells will be serum starved for 48 hours followed by replating
of cells at low density in serum and MTX. Colonies will be selected
and expanded for analysis of kinase activity against 40S ribosomal
subunits, and for coexpression of the two proteins. Further
controls will be performed for inhibition of protein association
with wortmannin and rapamycin.
e) Modification of the Eukaryotic Survival Assay for Library
Screening
An important part of the work required in creating a library for
use in eukaryotic cells will have been accomplished already, as the
EcoRI/XhoI directional cDNA produced by the Stratagene "cDNA
Synthesis Kit" can directly be inserted directionally into the
Stratagene Zap Express vector.
Methodology:
Steps 1 through 5 are parallel to those for the bacterial library
screening (above). 1--Again, the library is constructed as a
C-terminal fusion to mDHFR F[3]. F[3] (with no stop codon) will be
inserted in frame in Zap Express, followed by insertion of the
novel polylinkers allowing expression of the inserts in all three
reading frames (described above), and by the EcoRI/XhoI directional
cDNA. This bacteriophage library will be propagated and treated
with the Stratagene helper phage to excise a eukaryotic expression
phagemid vector (pBK-CMV) carrying the fusion inserts.
2--Cotransfection of the library and p70S6k-F[1,2] constructs in
eukaryotic cells: we will perform the screening in COS or 293
cells, as these are responsive to serum in activating the p70S6k
signaling pathway. Selection experiments will be performed as
described for the S6 test system above. 3--Propagation, isolation
and sequencing of the insert DNA will be undertaken. 4--The cloned
fusion proteins will be sized on SDS-PAGE by direct visualization
after 35S-Met/Cys labeling, or by Western blotting using a
commercial polyclonal antibody to mDHFR.
Generalization of the Strategy: The scheme for detecting partners
for the protein p70S6k can be applied to studies of any biochemical
pathway in any living organism. Such pathways may also be related
to disease processes. The disease-related pathway may be an
intrinsic process of cells in humans where a pathology arises from,
for instance mutation, deletion or under or over expression of a
gene. Alternatively the biochemical pathway may be one that is
specific to a pathogenic organism or the mechanism of host
invasion. In this case, component proteins of such processes may be
targets of a therapeutic strategy, such as development of drugs
that inhibit invasion by the organism or a component enzyme in a
biochemical pathway specific to the pathogenic organism.
Inflamatory diseases are a case in point that can concern both
examples. The protein-protein interactions that mediate the
adhesion of leukocytes to inflamed tissues are known to involve
such proteins as vascular cell adhesion molecule-1 (VCAM-1), and
certain cytokines such as IL-6 and IL-8 that are produced during
inflammation. However, many of the proteins involved in onset of
inflammatory response remain unknown; further, the intracellular
signaling pathways triggered by the extracellular associations are
poorly understood. The PCAs could be used in elucidation of the
mechanisms underlying the onset of inflammation, as well the
ensuing signaling. For example, signaling pathways associated with
inflammation, such as those mediated by IL-1, IL-6, IL-8 and tumor
necrosis have been studied in some detail and many direct and
downstream regulators are known. These regulators can be used as
starting point targets in a PCA screening to identify other
signalling or modulating proteins that could also be targets for
drug development.
There is an increased risk of infection by enteric pathogens in the
occurrence of the intestinal inflammation that characterizes
idiopathic intestinal diseases. There are two mechanisms which need
to be better understood here and which can be addressed by PCA:
i--the cellular mechanisms of inflammation as described above,
and
ii--the discovery of the specific cell-surface ligands which the
pathogenic organisms recognize and associate with. Secreted
proteins produced by the pathogen can bind to the basolateral
membrane of epithelial cells (as in the case in Yersinia
pseudotuberculosis infection) or be translocated into intestinal
epithelial cells (Salmonella infection), promoting infectivity
and/or physiological responses to the infection. However, in most
cases the interactions between the pathogenic protein and the
epithelial cells are unknown. Cell adhesion and nervous system
regeneration A related example in cell adhesion includes processes
involved in development and regeneration in the nervous system.
Cadherens are membrane proteins that mediates calcium dependent
cell-cell adhesion. To do so they need another class of cytoplasmic
proteins called cathenins. Those make a bridge between cadherins
and cytoskeleton. Cathenins are also regulate genes that control
differentiation-specific genes. For instance, the protein
B-cathenin can interact in certain situation with a transcription
factor (lef-1) and be translocated into the nucleus where it
constrains the number of genes transactivated by lef-1
(differentiation). This process is regulated by the Wnt signaling
pathway (homologs to the wingless pathway in drosophila) by
inactivation of GSK3B which permit degradation after of APC (a
cytoplasmic adapter protein). PCA strategies could be used to
identify novel proteins involved in the regulation of these
processes.
Proteins involved in viral integration processes are examples of
targets that could be tested for inhibitors using the PCA
strategies. Examples for the HIV virus include:
i) inhibition of integrase or the transport of the pre-integration
complex: protein Ma or vpr.
ii) Inhibition of the cell cycle in G2 by vpr (interaction by
cyclin B) causing induction of apoptosis.
iii) Inhibition of the interaction of gp160 (precursor of the
membrane proteins) with furine.
Accessory proteins of HIV as a therapeutic target:
i) Vpr: nuclear localizing sequence (target): interaction site of
vpr with phosphatasesA.
ii) vif: interaction with vimentin (cytoskeleton associated
protein).
ii) Vpu: Degradation of CD4 in the RE mediated by the cytoplasmic
tail of Vpu.
iii) net Myristoylation signal of Nef.
Example 3
Other Examples of Protein Fragment Complementation Assays
Other examples of assays are herein exemplified. The reason to
produce these assays is to provide alternative PCA strategies that
would be appropriate for specific protein association problems such
as studying equilibrium or kinetic aspects of assembly. Also, it is
possible that in certain contexts (for example, specific cell
types) or for certain applications, a specific PCA will not work
but an alternative one will. Further below are brief descriptions
of each other PCAs embodiments.
1) Glutathione-S-Transferase (GST) GST from the flat worm
Schistosoma japonicum is a small (28 kD), monomeric, soluble
protein that can be expressed in both prokaryotic and eukaryotic
cells. A high resolution crystal structure has been solved and
serves as a starting point for design of a PCA. A simple and
inexpensive colorimetric assay for GST activity has been developed
consisting of the reductive conjugation of reduced glutathione with
1-chloro-2,4-dinitrobenzene (CNDB), a brilliant yellow product. We
have designed a PCA based on similar structural criteria used to
develop the DHFR PCA using GCN4 leucine zippers as oligomerization
domains. Cotransformants of zipper-GST-fragment fusions are
expressed in E. coli on agar plates and colonies are transferred to
nitrocellulose paper. Detection of fragment complementation is
detected in an assay where a glutathione-CDNB reaction mixture is
applied as an aerosol on the nitrocellulose and colonies expressing
co-expressed fragments of GST are detected as yellow images. 2)
Green Fluorescent Protein (GFP) GFP from Aequorea victoria is
becoming one of the most popular protein markers for gene
expression. This is because the small, monomeric 238 amino-acids
protein is intrinsically fluorescent due to the presence of an
internal chromophore that results from the autocatalytic
cyclization of the polypeptide backbone between residues Ser65 and
Gly67 and oxidation of the--bond of Tyr66. The GFP chromophore
absorbs light optimally at 395 nm and possesses also a second
absorption maximum at 470 nm. This bi-specific absorption suggests
the existence of two low energy conformers of the chromophore whose
relative population depends on local environment of the
chromophore. A mutant Ser65Thr that eliminates isomerization
(single absorption maximum at 488 nm) results in a 4 to 6 times
more intense fluorescence than the wild type. Recently the
structure of GFP has been solved by two groups, making it now a
candidate for a structure-based PCA-design, which we have begun to
develop. As with the GST assay, we are doing all of our initial
development in E. coli with GCN4 leucine zipper-forming sequences
as oligomerization domains. Direct detection of fluorescence by
visual observation under broad spectrum UV light will be used. We
will also test this system in COS cells, selecting for
co-transfectants using fluorescence activated cell sorting (FACS).
3) Fire Fly Luciferase. Firefly luciferase is a 62 kDa protein
which catalyzes oxidation of the heterocycle luciferin. The product
possesses one of the highest quantum yields for bioluminescent
reactions: one photon is emitted for every oxidized luciferin
molecule. The structure of luciferase has recently been solved,
allowing for structure-based development of a PCA. As with our GST
assay, cells are grown on a nitrocellulose matrix. The addition of
the luciferin at the surface of the nitrocellulose permits it to
diffuse across the cytoplasmic membranes and trigger the
photoluminescent reaction. The detection is done immediately on a
photographic film. Luciferase is an ideal candidate for a PCA: the
detection assays are rapid, inexpensive, very sensitive, and
utilizes non-radioactive substrate that is available commercially.
The substrate of luciferase, luciferin, can diffuse across the
cytoplasmic membrane (under acidic pH), allowing the detection of
luciferase in intact cells. This enzyme is currently utilized as a
reporter gene in a variety of expression systems. The expression of
this protein has been well characterized in bacterial, mammalian,
and in plant cells, suggesting that it would provide a versatile
PCA. 4) Xanthine-guanine phosphoribosyl transferase (XGPRT) The E.
coli enzyme XGPRT converts xanthine to xanthine monophosphate
(XMP), a precursor of GMP. Because the mammalian enzyme
hypoxanthine-guanine phosphoribosyl transferase HGPRT can only use
hypoxanthine and guanine as substrates, the bacterial XGPRT can be
used as a dominant selection assay for a PCA for cells grown in the
presence of xanthine. Vectors expressing XGPRT confer the ability
of mammalian cells to grow in selective medium containing adenine,
xanthine, and mycophenolic acid. The function of mycophenolic acid
is to inhibit de novo synthesis of GMP by blocking the conversion
of IMP into XMP (Chapman A. B., (1983) Molec. & Cellul. Biol.
3, 1421-1429). The only GMP produced then come from the conversion
of xanthine into XMP, catalyzed by the bacterial XGPRT. As with
aminoglycoside phosphotransferase fragments of XGPRT can be
generated based on the known structure (See table 1.) using the
design-evolution strategy described above with fragments fused to
the GCN4 leucine zippers as a test oligomerization domains. The
complementary fusions are cotransfected and the proteins
transiently expressed in COS-7 cells, or stability expressed in CHO
cells, grown in the selective medium. In the case of CHO cells,
colonies are collected and sequentially re-cultured at increasing
concentrations of the selective compounds in order to enrich for
populations of cells that efficiently express the fusions at high
concentrations. 5) Adenosine deaminase Adenosine deaminase (ADA) is
present in minute quantities in virtually all mammalian cell.
Although it is not an essential enzyme for cell growth, ADA can be
used in a dominant selection assay. It is possible to establish
growth conditions in which the cells require ADA to survive. ADA
catalyzes the irreversible conversion of cytotoxic adenine
nucleosides to their respective nontoxic inosine analogues. By
adding cytotoxic concentrations of adenosine or cytotoxic adenosine
analogues such as 9-b-D-xylofuranosyladenine to the cells, ADA is
required for cell growth to detoxify the cytotoxic agent. Cells
that incorporate the ADA gene can then be selected for
amplification in the presence of low concentrations of
2'-deoxycoformycin, a tight-binding transition state analogue
inhibitor of ADA. ADA can then be used for a PCA based on cell
survival (Kaufman, R. J. et al. (1986) Proc. of the Nat. Acad. Sci.
(USA) 83, 3136-3140). As with the other systems described above,
fragments of ADA can be generated based on the known structure (See
table 1.) using the design-evolution strategy described above with
fragments fused to the GCN4 leucine zippers as a test
oligomerization domains. The complementary fusions are
cotransfected and the proteins transiently expressed in COS-7
cells, or stability expressed in CHO cells, grown in the selective
medium containing 2'-deoxycoformycin. In the case of CHO cells,
colonies are collected and sequentially re-cultured at increasing
concentrations of 2'-deoxycoformycin in order to enrich for
populations of cells that efficiently express the fusions at high
concentrations 6) Bleomycin binding protein (zeocin resistance
gene) Zeocin, a member of the bleomycin/phleomycin family of
antibiotics, is toxic to bacteria, fungi, plants, and mammalian
cells. The expression of the zeocin resistance gene confers
resistance to bleomycin/zeocin. The protein confers resistance by
binding to and sequestering the drug and thus preventing its
association and hydrolysis of DNA. Berdy, J. (1980) In Amino Acid
and Peptide Antibiotics, J. Berdy, ed. (Boca Raton, Fla.: CRC
Press), pp. 459-497; Mulsant, P., Tiraby, G., Kallerhoff, J., and
Perret, J. (1989 Somat. Cell. Mol. Genet. 14, 243-252). Bleomycin
binding protein (BBP) could then be used for a PCA based on cell
survival. As with the other systems described above, fragments of
ADA can be generated based on the known structure (See table 1.)
using the design-evolution strategy described above with fragments
fused to the GCN4 leucine zippers as a test oligomerization
domains. The BBP is a small (8 kD) dimer that binds to drugs via a
subunit interface binding site. For this reason, the design would
be somewhat different in that first, a single chain form of the
dimer would be generated by making a fusion of two BBP genes with a
short sequence coding for a simple polypeptide linker introduced
between the two subunits. Fragments in this case will be based on a
short sequence of one of the subunit modules, while the other
fragment will be composed of the remaining sequence of the subunit
plus the other subunit. Complementation and selection experiments
will be performed as described for the examples above using
bleomycin or zeocin as selective drugs. 7)
Hygromycin-B-phosphotransferase. The antibiotic hygromycin-B is an
aminocyclitol that inhibits protein synthesis by disrupting
translocation and promoting misreading. The E. coli enzyme
hygromycin-B-phosphotransferase detoxifies the cells by
phosphorylating Hygromycin-B. When expressed in mammalian cells,
hygromycin-B-phosphotransferase can confer resistance to
hygromycin-B (Gritz, L., and Davies, J. (1983) Gene 25, 179-188.).
The enzyme is a dominant selectable marker and could be used for a
PCA based on cell survival. While the structure of the enzyme is
not known it is suspected that this enzyme is homologous to
aminoglycoside kinase (Shaw, et al. (1993) Microbiol. Rev. 57,
138-163). It is therefore possible to use the combined
design/evolution strategy to produce a PCA with this enzyme and
perform dominant selection in mammalian cells with selection at
increasing concentrations of hygromycin B. 8) L-histidinol
NAD+oxydoreductase. The hisD gene of Salmonella typhimurium codes
for the L-histidinol NAD+oxydoreductase that converts histidinol to
histidine. Mammalian cells grown in media lacking histidine but
containing histidinol can be selected for expression of hisD
(Hartman, S. C., R. C. Mulligan (1988) Proc. of the Nat. Acad. Sci.
(USA) 85, 8047-8051). An additional advantage of using hisD in
dominant selection is that histidinol is itself toxic, inhibiting
the activity of endogenous histidyl-tRNA synthetase. Histidinol is
also inexpensive and readily permeates cells. The structure of
histidinol NAD+oxydoreductase is unknown and so development of a
PCA based on this enzyme is based entirely on the exonuclease
fragment/evolution strategy. The following Table list alternative
embodiments using other PCA reporters. Abbreviations in Table:
Type: D, dominant selection marker; R, recessive selection marker.
Structure: four letter codes=Protein Data Bank (PDB) entries; K,
known but not deposited in PDB; U, unknown. mono/oligo: M, monomer;
D, dimer; tetra, tetramer.
TABLE-US-00001 TABLE 1 A list of Other Potential PCA Reporter
Candidates A-Assays based on Dominant or Recessive Selection mono/
Enzyme Type Structure Size oligo Selection drugs/Conditions DHFR
R/D many 18 kD M methotrexate/trimethoprim Adenosine deaminase D/R
1ADD M Xyl-A or adenosine, alanosine, and 2'- deoxycoformycin
Thymidine kinase D/R 1KIN D gangcyclovene, HAT Mutant
hypoxanthine-guanine D 1HGM D HAT + thymidine kinase phosphoribosyl
transferase Thymidylate synthetase R 1NJE 35 kd M 2
fluorodeoxyuridine Xanthine-guanine D 1NUL mycophenolic acid with
phosphoribosyl transferase limiting xanthine Glutamine synthetase R
2LGS Asparagine synthetase R U B-aspartyl hydroxamate or albizin
Puromycin N-acetyltransferase D U 23 kD M puromycin Aminoglycoside
D K 35 kD M neomycin, G418, gentamycin phosphotransferase
Hygromycin B D U M hygromycin B phosphotransferase L-histidinol:
NAD + D U 46 kD M histidinol oxidoreductase Bleomycin binding
protein D K 8 kD D bleomycin/zeocin Cytosine methyl-transferase R/D
U 5-Azacytidine (5-aza-CR) and 5-aza-2'-deoxycytidine
O6-alkylguanine D 1ADN N-methyl-N-nitrosourea alkyltransferase
Glycinamide ribonucleotide R 1GRC 23.2 kD D
dideazatetrahydrofolate, transformylase minus purine Glycinamide
ribonucleotide R U 45.9 kD minus purine synthetase
Phosphoribosyl-aminoimidazole R U 36.7 kD minus purine synthetase
Formylglycinamide ribotide R U 141.4 kD M L-azaserine,
6-diazo-5-oxo- amidotransferase L-norleucine, minus purine
Phosphoribosyl-aminoimidazole R U 39.5 kD D minus purine
carboxylase Phosphoribosyl-aminoimidazole R U 57.3 kD minus purine
carboxamide formyltransferase Fatty acid Synthase R 272 kD D
cerulenin IMP dehydrogenase R 1AK5 55.4 kD Tetra mycophenolic acid
ii-Viral Plaque Assays Mono/ Enzyme Type Structure Size Oligo
Selection drugs/Conditions Thioredoxin D 1TDF 34.5 kD D Reverse
transcriptase D 3HVT Viral protease D B-Cell Death Assays Mono/
Enzyme Type Structure Size Oligo Selection drugs/Conditions
Cysteine protease: papain D 1STF 38.9 kD M inhibited by cystatin
Cysteine protease: caspase D 1CP3 17 kD + HeteroD inhibited by
DEVD-aldehyde 12 kD (can also by used in a fluorimetric or
colorimetric assay, in vitro) Metalloprotease: D 47.1 kD M
inhibited by methyl-ethyl carboxypeptidase succinic acid Serine
protease: proteinase K D 1PTK 30.6 kD M inhibited by serpins
Aspartic protease: pepsin D 1PSN 34.5 kD M inhibited by pepstatin A
(can also be used in an fluorimetric assay, in vitro) Lysozyme D
many 23.2 kD M inhibited by N- acetylglucosamine trisaccharide
RNAse D many 13.3 kD M inhibited by RNAse inhibitor DNAse D 1DNK
61.6 kD M inhibited by actin Phospholipase A2 D 1P2P 13.8 kD M/D
many inhibitors: bromophenacyl bromide, hexadecyl-
trifluoroethyl-glycero- phosphomethanol, bromoenol lactone, etc.
Phospholipase C D 1AH7 28 kD M many inhibitors: neomycin,
chelerythrine, U73122, etc. C-Colorimetric/Fluorimetric Assay Mono/
Enzyme Structure Size Oligo Selection drugs/Conditions
DT-Diaphorase (NAD(P)H- 1QRD 26 kD D NADPH-diaphorase stain,
inhibited [quinone acceptor] by dicumarol, Cibacron blue and
oxidoreductase) phenidione Note: can also be used in a cell death
assay (+nitrobenzimidazole, fo example). (NAD(P)H-[quinone
acceptor] isoform of 21 kD D NRH-diaphorase stain, inhibited by
oxidoreductase)-2 1QRD pentahydroxyflavone Thermophilic diaphorase
30 kD M NADH-diaphorase stain (Bacillus stearothermophilus)
Glutathione-S-transferase 1GNE 26 kD D production of a yellow
product by other the conjugation of glutathione with isoform an
aromatic substance, chloro of dinitrobenzene (CDNB) 28 kD
Luciferase 1LCI 62 kD M Fluorometric Green-fluorescent protein 1EMA
30 kD M Intrinsic fluorescence Chloramphenicol 1CLA 25 kD Tri
Fluorimetric: Bodipy acetyltransferase chloramphenicol Uricase 32
kD Tetra Fluorometric SEAP (secreted form of human 1AJA M CSPD
chemiluminescent substrate placental alkaline phosphatase)
B-Glucuronidase 1BHG 71 kD Tetra Histochemical, fluorometric or
spectrophotometric assays using various substrates such as X-GLUC.
D-Heteromeric Enzyme Strategies Tyrosinase 30 kD + Hetero
Colorimetric: synthesis of melanin 14 kD M + M
Example 4
Examples of Variants of PCA to Detect Multiple
Protein/Protein-DNA/Protein RNA/Protein-Drug Complexes
To this point specific examples have only been made of applications
of PCA to protein-pair interactions. However, it is possible to
apply PCA to multiprotein, protein-RNA, protein-DNA or
protein-small molecule interactions. There are two general schemes
for achieving such systems. Multi-subunit PCA: Two proteins need
not interact for a PCA signal to be observed; if a partner protein
or protein complex binds to two proteins simultaneously, it is
possible to detect such a three protein complex. A multusubunit PCA
is conceived with the example of herpes simplex virus thymidine
kinase (TK), a homodimer of 40 kD. In this conception, the TK
structure contains two well defined domains consisting of an
alpha/beta (residues 1-223) and an alpha-helical domain (224-374).
As a test system, we use the Rop1 dimer, a four helix bundle
homodimer. The two fragments of TK are extracted by PCR and
subcloned into the transient transfection vector pMT3, the first in
tandem to the Adenovisus major late promoter, tripartite leader 3'
to the first ATG, and the second downstream of a ECMV internal
initiation site. Restriction sites previously introduced between
the first and the last ATG are subcloned into BamHI/KpnI and
PstI/EcoRI cloning sites downstream of the two ATGs. These are used
to subclone PCR-generated fragments of the Rop1 subunits into two
different vectors. Subsequently Ltk-cells are cotransfected by
lipofection with the two plasmids and colonies of surviving cells
are serially selected in medium containing increasing
concentrations of HAT (hypoxanthine/aminopterin/thymidine). Cells
that express complementary fragments of TK fused to the four Rop1
will proliferate under this selective pressure, or otherwise die.
Specific examples of use of this concept would be in determining
constituents of multiprotein complexes that are formed transiently
or constitutively in cells.
The utility of PCA is not limited to detecting protein-protein
interactions, but can be adapted to detecting interactions of
proteins with DNA, RNA, or small molecules. In this conception, two
proteins are fused to PCA complementary fragments, but the two
proteins do not interact with each other. The interaction must be
triggered by a third entity, which can be any molecule that will
simultaneously bind to the two proteins or induce an interaction
between the two proteins by causing a conformational change in one
or both of the partners. Two examples have been demonstrated in our
lab using the mDHFR PCA in E. coli. In the first case a natural
product, the immunosuppressant drug rapamycin, is used to induce an
interaction between its receptor FKBP12 and a partner protein mTOR
(mammalian Target of Rapamycin). We detect this by cotransformation
of DHFR fragments fused to FKBP or mTOR into E. coli grown in the
presence or absence of trimethoprim (as described above) and
rapamycin (0-10 nM). We have demonstrated that support of growth as
detected by colony formation is completely dependent on the
addition of rapamycin, suggesting that the mDHFR PCA is detecting a
rapamycin-induced assembly of a FKBP12-mTOR and subsequent
reconstitution of DHFR activity. This is one example of a use of
the PCA strategy to test for small molecules that can induce
interactions between proteins. General applications could be made
to therapeutic development, in the form screening small molecule
combinatorial compound libraries for molecules that induce
interactions between proteins, that may inhibit the activities of
either or both of the proteins, or activate specific cellular
processes that are initiated by other events, such as growth
factor-mediated receptor dimerization. The discovery of such small
molecules could lead to the development of orally available drugs
for the treatment of a broad spectrum of human diseases.
Another example of an induced interaction we have studied with the
DHFR PCA is the interaction of the oncogene GTPase p21 ras and its
direct downstream target, the serine/threonine kinase raf. This
interaction only occurs when the GTPase is in the GTP-bound form,
whereas turnover of GTP to GDP leads to release of the complex. As
with the FKBP-mTOR complex, we have demonstrated this induced
interaction in E. coli. PCA could be used in a general way to study
such induced interactions, and to screen for compounds that release
or prevent these interactions in pathological states. The ras-raf
interaction itself could be a target of therapeutic intervention.
Oncogenic forms of ras consist of mutants that are incapable of
turning over GTP and therefore remain continuously associated with
activated ras. This leads to a constitutive uncontrolled growth
signal that results, in part, in oncogenesis. The identification of
compounds that inhibit this process, by PCA, would be of value in
broad treatment of cancers. Other examples of multimolecular
applications of PCA could include identification of novel DNA or
RNA binding proteins. In its simplest conception one uses a known
DNA or RNA binding motifs, for instance a retinoic acid receptor
zinc finger, or a simple RNA binding protein such as IF-1,
respectively. One half of the PCA consists of the DNA or RNA
protein binding domain fused to one of the PCA fragments (control
fragment). The complementary fragment is fused to a cDNA library. A
third entity, the gene coding for a sequence containing an element
known to bind to the control protein, and then a second putative or
known regulatory element is coded for after this sequence. A test
system consists of tat/tar elements that control elongation in
transcription/translation of HIV genes. An example application
would be identification of tat binding elements that have been
proposed to exist in eukaryotic genomes and may regulate genes in
the same or similar way to that of HIV genes. (SenGupta D. J. et
al. (1996) Proc. Natl. Acad. USA 6, 8496-8501).
Example 5
Examples of PCA Applications to Drug Screening: Screening
Combinatorial Libraries of Compounds for Those that Inhibit or
Induce Protein-Protein/Protein-rna/Protein-DNA Complexes
A) Drug Screening.
Screening combinatorial libraries of compounds for those that
inhibit or induce protein-protein/protein-rna/protein-DNA
complexes. The PCA strategy can be directly applied to identifying
potentially therapeutic molecules contained in combinatorial
libraries of organic molecules. It is possible to perform high
throughput screening of such libraries to screen for compounds that
will inhibit or induce protein-protein interactions or
protein-DNA/RNA interactions (as discussed above). In addition it
is also possible to screen for compounds that inhibit enzymes whose
substrates are other proteins DNA, RNA or carbohydrates, as
discussed below. In this application, proteins that
interact/protein substrate pairs, or control DNA/RNA binding
protein-enzyme pairs are fused to PCA complementary fragments and
plasmids harboring these pairs are transformed/transfected into a
cell, along with any third DNA or RNA element as the case requires.
Transformed/transacted cells are grown liquid culture in multiwell
plates where each well is inoculated with a single compound from an
array of combinatorially synthesized compounds. A readout of a
response depends on the effect of a compound. If the compound
inhibits a protein interaction, there is a negative response (no
PCA signal is the positive response). If the compound induces a
protein interaction, the response is a positive PCA signal.
Controls for non-specific effects of compounds include: 1)
demonstration that the compound does not effect the PCA enzyme
itself (test against cells transfected with the wild-type intact
enzyme used as the PCA probe) and in the case of a cell survival
assay, that the compound is not toxic to the cells that have not
been transformed/transfected. As well as providing a high
throughput assay for biological activity of compounds, PCA also
offers the advantage over in vitro assays that it is a test for
cell membrane permeability of active compounds. Specific
demonstrated examples of PCA for drug screening in our laboratory
include the application of DHFR PCA in E. coli to detecting
compounds that inhibit therapeutically relevant targets. These
include Bax/Bcl2 fkbp12/tor ras/raf, carboxyl terminal dimerization
domain of HIV-1 capsid protein, IkB kinase IKK-1 and IKK-2
dimerization domains (leucine zippers and helix-loop-helix
domains). In each case, the two proteins are subcloned 5' upstream
of either F[1,2] or F[3] as described above. Plasmids harboring the
complementary fragments are cotransformed into BL21 cells. Colonies
from minimal medium plates containing IPTG and trimethoprim are
picked, and grown in liquid medium under the same selective
conditions and frozen stocks made. For a single screening cycle, a
priming overnight culture is grown from frozen stocks in LB medium.
A selective minimal medium containing trimethoprim, ampicillin,
IPTG is aliquated at 25 ml into each well of a 384 well plate. Each
well is then inoculated with 1 ul of an individual sample from a
compound array (ArQule Inc.) to give a final concentration of 10
uM. Each well is then inoculated with 2 ml of overnight culture and
plates are incubated in a specially adapted shaker bath at 37 C. At
2 hour intervals, plates are read on an optical absorption
spectroscopic plate reader coupled to a PC and spreadsheet software
at 600 nm (scattering) for a period of 8 hours. Rates of growth are
calculated from individual time readings for each well and compared
to a standard curve. A "hit" is defined as a case where an
individual compound reduces the rate of growth to less than the 95%
confidence interval based on the standard deviation for growth
rates observed in all of the wells within the test plate. "Near
hits" are defined as those cases where growth rates are within the
95% confidence interval. For each of the hits or near hits, the
following controls are then performed: The same experiment is
performed with BL21 cells that are transformed with empty vector
(and no trimethoprim), with vector harboring the full length mDHFR
gene, or with cotransfected cells where protein expression is not
induced by IPTG. If in all of these cases the compound has no
effect, it can be concluded that it is specifically disrupting the
protein-protein interaction being tested. Such validated hits or
near hits are then retested to establish a dose-response curve for
the individual compound, with concentrations varying from 1 pM up
to 1 .mu.M by orders of magnitude of 10. The PCA strategy for
compound screening can also be applied in the multiprotein
protein-RNA/DNA cases as described above, and can easily be adapted
to the DHFR or any other PCA in E. coli or in yeast versions of the
same PCAs. Such screening can also be applied to enzymes whose
targets are other proteins or nucleic acids for known
enzyme/substrate pairs or to novel enzyme substrate pairs
identified as described below.
Proteins involved in viral integration processes are examples of
targets that could be tested for inhibitors using the PCA
strategies. Examples for the HIV virus include:
i) inhibition of integrase or the transport of the pre-integration
complex: protein Ma or vpr
ii) Inhibition of the cell cycle in G2 by vpr (interaction by
cyclin B) causing induction of apoptosis.
iii) Inhibition of the interaction of gp160 (precursor of the
membrane proteins) with furine.
Accessory proteins of HIV as a therapeutic target:
i) Vpr: nuclear localizing sequence (target): interaction site of
vpr with phosphatasesA.
ii) vif: interaction with vimentin (cytoskeleton associated
protein).
ii) Vpu: Degradation of CD4 in the RE mediated by the cytoplasmic
tail of Vpu.
iii) net Myristoylation signal of Nef.
Other general targets for drug screening could include proteins
linked neurodegenerative diseases, such as to alpha-synuclein. This
protein has been linked to early onset of Parkinson disease and it
is present also implicated in Alzheimer disease. There is also
.beta.-amyloid proteins, linked to Alzheimers disease.
An example of protein-carbohydrate interactions that would be a
target for drug screening includes the selectins that are generally
implicated in inflammation. These cell surface glycoproteins are
directly involved in diapedesis.
A number of tumor suppressor genes whose actions are mediated by
protein-protein interactions could be screened for potential
anti-cancer compounds. These include PTEN, a tumor suppressor
directly involved in the formation of harmatomas. It is also
involved in inherited breast and thyroid cancer. Other interesting
tumor suppressor genes include p53, Rb and BARC1.
Example 6
Examples of Applications the PCA Strategy to Detect
Enzyme/Substrate Interactions
The examples described above are used for identifying novel
molecular interactions involving molecules that merely bind to each
other. However detecting the substrates of enzymes is also fully
compatible with the PCA strategy as shown below:
i) Enzymes that form tight complexes or with protein substrates or
induce efficient PCA fragment assembly or
ii) Mutant enzymes that bind tightly to substrate but do not
undergo product release because of mutations residues involved in
nucleophilic attack and/or product release (substrate
trapping).
Enzymes may form tight complexes with their substrates (Kd
.about.1-10 .mu.M). In these cases PCA may be efficient enough to
detect such interactions. However, even if this is not true, PCA
may work to detect weaker interactions. Generally, if the rate of
catalysis and product release is slower than the rate of
folding--reassembly of the PCA complementary fragments, effectively
irreversible folding and reconstitution of the PCA reporter
activity will have occurred. Therefore, even if the enzyme and
substrate are no longer interacting, the PCA signal is detected.
Therefore, the detection of novel enzyme substrates using PCA may
be possible, independent of effective substrate Kd or rate of
product release. In cases where product release is much faster than
PCA fragment assembly/folding and alternative approach is provided
by generating "substrate trapping" mutants of the test enzyme. An
example of this approach applied to the protein tyrosine
phosphatase PTP1B, where substrate trapping mutants have been
generated by mutating the nucleophilic aspartate 181 to alanine
rendering the enzyme catalyticly dead, but capable of forming tight
complexes with a known substrate, the EGF receptor and other
unknown proteins (Flint, A. J. et al. (1996) Proc. Natl. Acad. USA
941680-1685). An application of using PCA to screen for interacting
partners of PTP1B is given as follows. We use the aminoglycoside
kinase (AK)-based PCA in transiently transfected COS or 293 cells.
The substrate trap mutant catalytic domain of PTP1B is fused to
N-terminal complementary fragment of AK, while a C-terminal fusion
of the other AK fragment is made to a cDNA library. Cells are
cotransfected with complementary AK pairs and grown in selective
concentrations of G418. After 72 hours, colonies of surviving cells
are picked and in situ PCR is performed using primers designed to
anneal to 3' and 5' flanking regions of the cDNA coding region. PCR
amplified products are then 5' sequenced to identify the gene.
Enzyme inhibitors Screening combinatorial libraries of compounds
for those that inhibit enzyme-PROTEIN substrate complexes either
with:
i) Enzymes that form tight complexes with protein substrates or
ii) Mutant enzymes that bind tightly to substrate but do not
undergo product release because of the mutation.
Example 7
Applications of the PCA Strategy to Protein
Engineering/Evolution
The PCA strategy can be used to generate peptides or proteins with
novel binding properties that may have therapeutic value, as is
done today with phage display technology. It is also possible to
develop enzymes with novel substrate or physical properties for
industrial enzyme development. Two detailed examples of the
application of the PCA strategy to these ends are given below, with
additional applications listed below.
1) Selection of high-affinity, heterodimerizing leucine zipper
sequences (J. Pelletier, K. Arndt, A. Plueckthun and S. Michnick,
manuscript in preparation). The mDHFR PCA, described above, was
used in a scheme for the selection of efficiently heterodimerizing,
designed leucine zippers. It has been proposed that the formation
of salt bridges between positively and negatively charged residues
at complementing "e" and "g" positions is important in stabilizing
leucine zipper formation, though this view has been contested. In
order to help define the importance of salt-bridge formation at the
e and g positions, two leucine zipper libraries were built. Both
are based on the GCN4 leucine zipper sequence, but contain sequence
information specific to either Jun or Fos zippers in order to
create heterodimerizing pairs. As well, the e-1 to e-4 and g-1 to
g-4 positions in each library were randomized to code for
positively or negatively charged residues, or neutral polar
residues. These libraries were amplified by PCR and subcloned into
the Z-F[1,2] or Z-F[3] constructs (described above) from which the
GCN4 zipper sequences had been removed. The bacterial mDHFR PCA
selection was performed on selective solid media, as described
earlier. Colonies were picked and sequenced; sequence analysis
reveals that the distribution of charged or neutral residues at e-g
pairs is not random, but is biased toward pairing of opposite
charges, or pairing of a charged with a neutral residue, rather
than same-charge pairing (see FIG. 7). We reasoned that better
zipper pairing should lead to an increase in efficiency of
DHFR-fragment complementation, resulting in faster bacterial
doubling times (see Table 1 in the mDHFR PCA description), and
undertook a selection/enrichment of the novel zippers relative to
GCN4, as follows. The designed zipper libraries, expressed as
N-terminal fusions to the DHFR F[1,2] or F[3:I114A], were
cotransformed, clones were picked, propagated and mixed in
selective liquid culture, and the mix was added in a 1:1 000 000
ratio to clone Z-F[1,2]+Z-F[3:I114 A] (original GCN4 leucine
zippers). The mixture was propagated in selective liquid culture
over multiple passages. Restriction analysis shows that within 4
passages, the population of GCN4-expressing bacteria is diminishing
relative to the novel zipper sequences (data not shown), indicating
that some of the designed zipper-containing clones are propagated
at a higher rate than those containing GCN4. Bacteria from later
passages were plated on selective medium, and individual clones
sequenced to reveal the identity of the most successful designed
zipper pairs (data not shown). 2) Application of PCA to enzyme
function and design PCA Development: Adenosine deaminase (ADA)
meets all of the criteria for a PCA listed above. ADA is a small
(.about.40 kD), and easily purified monomeric zinc metallo-enzyme
and the structure of murine ADA has been resolved. Several in vitro
ADA activity assays have been developed, involving UV
spectrophotometry and stopped-flow fluorimetry. E. coli ADA
catalyzes the irreversible conversion of cytotoxic adenine
nucleosides to non-toxic inosines.
Eukaryotic or prokaryotic cells propagated in the presence of
cytotoxic concentrations of adenosine or adenosine analogs require
ADA to detoxify these compounds. This is the basis of a
dominant-selection strategy used to select for cells expressing a
specific gene in mammalian cells. The ADA gene has also been
expressed in SF3834 E. coli cells which lack a gene coding for
endogenous ADA. When the gene coding for ADA is introduced into
ADA--bacterial DNA, those cells that express ADA are able to
survive high concentrations of added adenosine; those that do not,
die. This forms the basis of an in vivo ADA activity assay.
We chose ADA, principally because it can be used as a dominant
selective marker in mammalian and bacterial cells where the gene
has been knocked out. The reason we choose dominant selective genes
is because in screening for novel protein-protein interactions,
particularly testing for interactions of a known protein against a
library of millions of independent clones, selection serves to
filter for cells that may show a positive response for reasons
having nothing to do with a specific protein-protein interaction.
We will use three test systems of interacting proteins including
leucine zipper-forming sequences, the proteins raf and p21 and the
induced oligomerization system, FK506 binding protein (FKBP) and
mTOR that interact through the macrocyclic immuno-suppressant
compound rapamycin. For all of these systems, we will construct E.
coli and mammalian transient transfection plasmids and subclone the
test proteins as fusions to ADA complementary fragments. The
primary assay will be survival of SF3834 E. coli cells that have
been transformed with the complementary ADA fragments fused to the
test oligomerization proteins in the presence of toxic
concentrations of adenosine. We will then purify fusion proteins
from colonies of and perform in vitro assays of ADA activity as
described below. The utility of the ADA PCA as a method to identify
novel proteins that interact with a test bait will be performed in
mammalian COS-7 and HEK-293T cells transiently transfected with
FKBP fused to one of the ADA fragments and the other fragment fused
to a cDNA library from normal human spleen containing 10.sup.6
independent clones. As with the E. coli assay, cells that survive
in a medium containing toxic concentrations of ADA is collected and
isolated plasmids will be tested to identify the gene for the
interacting protein by PCR amplification and chain
propagation-termination techniques.
Structural motifs required for protein function: Determination of
the structural elements required for the enzymatic function of ADA
are investigated through alteration of the structures of the enzyme
fragments. At first, ADA is cut into two separate domains--one
responsible for substrate binding (residues 1-210) and one
responsible for catalysis (residues 211-352). These separate pieces
will be attached to known assembly domains, such as leucine zippers
(see example 1 above). Reassembly will restore activity which will
be assessed through detailed in vitro kinetic analysis of the
binding and catalytic properties of the re-assembled enzyme, using
UV spectrophotometry and stopped-flow fluorimetry to observe the
enzymatic reactions. This system will provide another handle on the
manipulation of enzyme activity that will afford a powerful tool
for enzymatic mechanism study. For example, the difference in the
kinetic behaviour of the reassembled enzyme on mixing with the
substrate, compared to enzyme reassembled in presence of substrate
(where substrate may already be bound by binding domain) will allow
sophisticated level of study of importance of binding energy to
catalysis. Subsequent point mutations to the functional or assembly
domains of the proteins will then allow a very subtle perturbation
and detailed quantification of the relationship of binding energy
to catalysis. This precise control over the structure and assembly
of separate functional domains of the enzyme will permit very
sophisticated enzymatic structure function studies, the definition
of structural motifs and an understanding of their role in
catalysis. Novel protein catalyst design: The detailed knowledge of
the enzyme mechanism gained through determination of the structural
requirements for catalysis will then be exploited through the
combination of these functional "building blocks" with the
functional motifs responsible for substrate binding and catalysis
in other enzymes, allowing the generation of novel protein
catalysts. For example, the catalytic motif from ADA is modified to
a cytidine-binding motif, creating a novel enzyme with potentially
useful catalytic properties. The activity of these novel enzymes
can easily be assessed through in vivo assays similar to that of
the PCA system, or through in vitro activity assays. Furthermore,
the detailed mechanistic investigation of the resulting enzymes
possible with this system will permit the rational design of each
subsequent generation of catalysts.
Example 8
Examples of Applications the PCA Strategy to Detect Molecular
Interactions in Whole Organisms
It is a logical extension of the descriptions of PCA applications
above to the utility of these techniques in whole model organisms
such as drosophila, nematodes, zebra fish and puffer fish, as
examples. The sole differences with other listed examples is that
vectors used would need to be different (for example retroviral
vectors) and that any substrates needed by the PCA would need to be
bioavailable, or detection would need to be performed in situ.
Example 9
Examples of Applications of the PCA Strategy to Gene Therapy
Another important embodiment of the invention is to provide a means
and method for gene therapy of mammalian disease. Of particular
interest is the use of PCA therapeutic for treatment of cancer. In
one embodiment of said PCA gene therapy, a PCA is developed
employing fragments (modular protein units) derived from a protein
toxin for example: Pseudomonas exotoxin, Diptheria toxin and the
plant toxin gelonin, or other like molecules. For therapy of breast
cancer for example, first a mammalian, retroviral, adenoviral, or
eukaryotic artificial chromosomal (EAC's) genetic construct is
prepared that introduces one fragment of the selected toxin under
the control of the promoter for expression of the erbB2 oncogene.
Its is well known that the erbB2 oncogene is overexpressed in
breast cancer and adenocarcinoma cells (D. J. Slamon et. al.,
Science, 1989, 244, 707). The HER2/neu (c-erbB-2) proto-oncogene
encodes a sub-class 1 185-kDa transmembrane protein tyrosine kinase
growth factor receptor, p185.sup.HER2. Also, the human erbB2
oncogene is located on chromosome 17, region q21 and comprises
4,480 base pairs and p185.sup.HER2 serves as a receptor for a
30-kDa glycoprotein growth factor secreted by human breast cancer
cell lines (R. Lupu et. Al., Science, 1990, 249, 1152).
The transgene is introduced `in vivo` or `ex-vivo` into target
cells employing methods known by those skilled in the art e.g.
homologous recombination to insert transgene into locus of interest
via retroviral, adenoviral or EAC's. A second genetic construct
comprising a fusion gene containing a target DNA that encodes an
interacting protein that interacts with erbB2 oncogene discovered
by the PCA process described in this invention and the "second"
fragment of the toxin molecule. This construct is delivered to the
patient by methods known in the art for example as shown in U.S.
Pat. Nos. 5,399,346 and 5,585,237 whose entire contents are
incorporated by reference herein. Transgene expression of the erbB2
oncogene-toxin fragment described will now be under the control of
the constitutive oncogene promoter. Proliferating tumor cells will
thus produce one piece of the toxin attached as a fusion to the
erbB2 oncogene. In the presence of the second genetic construct
expressing the PCA discovered interacting erbB2 oncogene
"interacting protein-toxin fragment" construct then: erbB2
oncogene-toxin fragmentA: interacting protein-toxin fragment B will
be created and induce death of target tumor cells through creation
of an active toxin through Protein Fragment Complementation and
thus provide an efficacious and efficient therapy of said
disease.
This can be extended to other diseases and other toxins employing
techniques described and embodied in this invention.
Example 10
Examples of Applications the PCA Strategy to Detect Molecular
Interactions In Vitro
Any of the PCA strategies described above could be adapted to in
vitro detection. Unlike the in vivo PCAs however, detection would
be performed with purified PCA fragment-fusion proteins. Such uses
of PCA have the potential for use in diagnostic kits. For example
the test DHFR assay described above where the interacting domains
are FKBP12 and TOR could be used as a diagnostic test for rapamycin
concentrations for use in monitoring dosage in patients treated
with this drug. As shown above, the instant invention provides: 1)
Allow for the detection of protein-protein interactions in vivo or
in vitro. 2) Allow for the detection of protein-protein
interactions in appropriate contexts, such as within a specific
organism, cell type, cellular compartment, or organelle. 3) Allow
for the detection of induced versus constitutive protein-protein
interactions (such as by a cell growth or inhibitory factor). 4) To
be able to distinguish specific-versus non-specific protein-protein
interactions by controlling the sensitivity of the assay. 5) Allow
for the detection of the kinetics of protein assembly in cells. 6)
Allow for screening of cDNA libraries for protein-protein
interactions.
Further aspects of the invention can be demonstrated by identifying
novel interactions with the enzyme p70S6k, to determine its'
regulation and how separate signaling cascades converge on this
enzyme.
The PCA method is particularly useful for detection of the kinetics
of protein assembly in cells. The kinetics of protein assembly can
be determined using fluorescent protein systems.
In a further embodiment of the invention, PCA can be used for drug
screening. The techniques of PCA are used to screen for drugs that
block specific biochemical pathways in cells allowing for a
carefully targeted and controlled method for identifying products
that have useful pharmacological properties.
Although the present invention has been described herein above by
way of preferred embodiments thereof, it can be modified, without
departing from the spirit and nature of the subject invention as
defined in the appended claims.
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Abbreviations: PCA, Protein-fragment Complementation Assay; mDHFR,
murine dihydrofolate reductase; hDHFR, human dihydrofolate
reductase; Z-F[1,2], GCN4 leucine zipper-mDHFR fragment[1,2]; USPS,
ubiquitiN-based split-protein sensor; IPTG,
isopropyl-b-D-thiogalactopyranoside; PMSF, phenylmethylsulfonyl
fluoride; SDS-PAGE, SDS polyacrylamide gel electrophoresis.
* * * * *